Mitochondria regulate Ca2+ wave initiation and inositol trisphosphate signal transduction in oligodendrocyte progenitors

Mitochondria in oligodendrocyte progenitor cells (OPs) take up and release cytosolic Ca2+ during agonist-evoked Ca2+ waves, but it is not clear whether or how they regulate Ca2+ signaling in OPs. We asked whether mitochondria play an active role during agonist-evoked Ca2+ release from intracellular stores. Ca2+ puffs, wave initiation, and wave propagation were measured in fluo-4 loaded OP processes using linescan confocal microscopy. Mitochondrial depolarization, measured by tetramethyl rhodamine ethyl ester (TMRE) fluorescence, accompanied Ca2+ puffs and waves. In addition, waves initiated only where mitochondria were localized. To determine whether energized mitochondria were necessary for wave generation, we blocked mitochondrial function with the electron transport chain inhibitor antimycin A (AA) in combination with oligomycin. AA decreased wave speed and puff probability. These effects were not due to global changes in ATP. We found that AA increased cytosolic Ca2+, markedly reduced agonist-evoked inositol trisphosphate (IP3) production, and also enhanced phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the Ca2+ dependent protein gelsolin. Thus, the reduction in puff probability and wave speed after AA treatment may be explained by competition for PIP2 between phospholipase C and gelsolin. Energized mitochondria and low cytosolic Ca2+ concentration may be required to maintain PIP2, a substrate for IP3 signal transduction.     

Characterization of elementary Ca2+ release signals in NGF-differentiated PC12 cells and hippocampal neurons

Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.     

Role of elementary Ca(2+) puffs in generating repetitive Ca(2+) oscillations

Inositol (1,4,5)-trisphosphate (IP(3)) liberates intracellular Ca(2+) both as localized 'puffs' and as repetitive waves that encode information in a frequency-dependent manner. Using video-rate confocal imaging, together with photorelease of IP(3) in Xenopus oocytes, we investigated the roles of puffs in determining the periodicity of global Ca(2+) waves. Wave frequency is not delimited solely by cyclical recovery of the cell's ability to support wave propagation, but further involves sensitization of Ca(2+)-induced Ca(2+) release by progressive increases in puff frequency and amplitude at numerous sites during the interwave period, and accumulation of pacemaker Ca(2+), allowing a puff at a 'focal' site to trigger a subsequent wave. These specific 'focal' sites, distinguished by their higher sensitivity to IP(3) and close apposition to neighboring puff sites, preferentially entrain both the temporal frequency and spatial directionality of Ca(2+) waves. Although summation of activity from many stochastic puff sites promotes the generation of regularly periodic global Ca(2+) signals, the properties of individual Ca(2+) puffs control the kinetics of Ca(2+) spiking and the (higher) frequency of subcellular spikes in their local microdomain.     

Subcellular distribution of cytosolic Ca2+ in isolated rat hepatocyte couplets: evaluation using confocal microscopy.

Ca2+ agonists induce Ca2+ waves and other non-uniform Ca2+ patterns in the cytosol of epithelial cells. To define subcellular Ca2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DiOC6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca2+ were made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca2+ signals generally began at the basal pole of the hepatocytes, then spread to the canaliculus at average speeds of 80 micron/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca2+ waves radiate in a basal-to-apical direction in this cell type.     

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

Multi-dimensional resolution of elementary Ca2+ signals by simultaneous multi-focal imaging

Elementary events such as puffs and sparks are cytosolic microdomains of Ca2+ from which cellular Ca2+ signals are constructed. Because of the tight localization and fast kinetics of elementary events, imaging studies have been hindered by instrumental limitations of confocal and deconvolution fluorescence microscopy which necessitate compromises between spatial and temporal resolution. Here, we describe a novel, yet simple 'multi-focal' fluorescence microscopy system that employs three high-speed cameras focused at different axial depths to enable 4-dimensional imaging with millisecond resolution. We demonstrate the utility of this system for studies of puffs in Xenopus oocytes by mapping the axial distribution of puff sites, by obtaining measurements of puff amplitudes undistorted by focus error, and by deriving deblurred images that reveal novel sub-micron jumps of Ca2+ release sites.     

Regulatory and spatial aspects of inositol trisphosphate-mediated calcium signals

Hormones that act to release Ca2+ from intracellular stores initiate a signaling cascade that culminates in the production of inositol 1,4,5-trisphosphate (InsP3). The Ca2+ response mediated by InsP3 is not a sustained increase in the cytosolic Ca2+ concentration, but rather a series of periodic spikes that manifest as waves in larger cells. In vitro studies have determined that the key positive feedback parameter driving spikes and waves is a highly localized direct Ca(2+)-activation of InsP3-gated Ca2+ channels. Advances in fluorescent Ca2+ imaging have facilitated the resolution of individual positive feedback units. These studies have revealed that there are several modes of channel coupling underlying global Ca2+ signals; single channel openings or Ca2+ "blips," synchronized clusters of channels or Ca2+ "puffs," and cell wide calcium waves. It appears that the channel clusters that produce Ca2+ puffs are synchronized by the highly localized positive feedback that was predicted by the in vitro studies of channel regulation. Localization of InsP3-induced Ca2+ signals has been shown to be important for activation of several cellular processes including uni-directional salt flow and mitochondrial activation.     

Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes

The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells.     

Transformation of local Ca2+ spikes to global Ca2+ transients: the combinatorial roles of multiple Ca2+ releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.     

The changes in Ca2+ sparks associated with measured modifications of intra-store Ca2+ concentration in skeletal muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 microM on average in 10 experiments) and a high value (433 microM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The "strength" of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.     

Mechanisms of subcellular cytosolic Ca2+ signaling evoked by stimulation of the vasopressin V1a receptor.

Receptor activation may result in distinct subcellular patterns of Ca2+ release. To define the subcellular distribution of Ca2+i signals induced by stimulation of the vasopressin V1a receptor, we expressed the cloned receptor in Xenopus oocytes. Oocytes were then loaded with fluo-3 and observed using confocal microscopy. Vasopressin induced a single concentric wave of increased Ca2+ that radiated inward from the plasma membrane. With submaximal stimulation, however, regions of the Ca2+ wave spontaneously reorganized into repetitive (oscillatory) waves. Focal stimulation of a small part of the plasma membrane resulted in a Ca2+ wave which began at the point of stimulation, radiated toward the center of the cell, then reorganized into multiple foci of repetitive, colliding waves and spirals of increased Ca2+i. The pattern of Ca2+ signaling induced by focal or global stimulation was not altered in Ca(2+)-free medium, although signals did not propagate as fast. Finally, subcellular Ca2+ signaling patterns induced by vasopressin were inhibited by caffeine, while neither vasopressin nor microinjection of inositol trisphosphate blocked caffeine-induced increases in cytosolic Ca2+. Thus, stimulation of the V1a receptor in this cell system induces a complex pattern of Ca2+ signaling which is influenced by (1) the magnitude of the stimulus, (2) the distribution of the surface receptors that are stimulated, and (3) mobilization of Ca2+ from the extracellular space as well as from two distinct endogenous Ca2+ pools. The manner in which a single type of receptor is activated may represent an important potential mechanism for subcellular Ca2+i signaling.     

Initiation of IP(3)-mediated Ca(2+) waves in Xenopus oocytes.

Inositol (1,4,5)-trisphosphate (IP(3)) evokes Ca(2+) liberation in Xenopus oocytes as elementary events (Ca(2+) puffs) that become coupled to propagate Ca(2+) waves with increasing [IP(3)]. To investigate this transition between local and global Ca(2+) signaling, we developed an optical method for evoking rapid subcellular Ca(2+) elevations, while independently photoreleasing IP(3) and simultaneously recording confocal Ca(2+) images. Focal Ca(2+) elevations triggered waves within 100 ms of photoreleasing IP(3), compared with latencies of seconds following photorelease of IP(3) alone. Wave velocity varied with [IP(3)] but was independent of time after photorelease of IP(3), indicating that delayed wave initiation did not involve slow binding of IP(3) to its receptors. The amount of Ca(2+) required to trigger a wave was approximately 10-fold greater than the average size of puffs, and puffs showed no progressive increase in magnitude before waves initiated. Instead, Ca(2+) puffs contributed to a slow rise in basal free [Ca(2+)], which further increased puff frequency and sensitized IP(3) receptors so that individual events then triggered waves. Because the wave threshold is much greater than the size of the elementary puff, cells can employ both local and global signaling mechanisms, and the summation of stochastic behavior of elementary events allows generation of reproducible periodic waves.     

Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal

Using single-cell ratio imaging of Fura-2-loaded neutrophils, we demonstrate that the heterogeneity and asynchrony of the oxidase response originates from variability in the timing and magnitude of the cytosolic free Ca2+ signal. The Ca2+ signals from individual cells could be classified into four types: (a) type 1, a transient rise in Ca2+ occurring within 6 s; (b) type 2, an oscillating cytosolic free Ca2+; (c) type 3, a latent Ca2+ transient significantly delayed (21-56 s); and (d) type 4, no significant Ca2+ rise. These response types accounted for approximately 41%, 15%, 26% and 18% of the population respectively for stimulation with 1 microM f-met-leu-phe peptide (n = 27) and 52.5%, 15%, 11.5% and 21% respectively for 0.1 microM f-met-leu-phe peptide (n = 52). The oxidase in neutrophils in which the cytosolic free Ca2+ concentration rose to greater than 250 nM always became activated. In the presence of extracellular Ca2+, cytosolic Ca2+ rose uniformly throughout the cell, whereas in the absence of extracellular Ca2+, a localised Ca2+ 'cloud' was observed in approximately 30% of cells. A localised activation of the oxidase accompanied the presence of the Ca2+ 'cloud' when the 250 nM Ca2+ threshold was exceeded. The data presented here therefore demonstrate a tight coupling in individual neutrophils between an elevation in cytosolic free Ca2+ above a threshold of 250 nM and activation of the oxidase.     

Endoplasmic reticulum Ca2+ store: regulation of Ca2+ release and reuptake by intracellular and extracellular Ca2+ in pancreatic acinar cells

We investigated the effect of cytosolic and extracellular Ca2+ on Ca2+ signals in pancreatic acinar cells by measuring Ca2+ concentration in the cytosol([Ca2+]c) and in the lumen of the ER([Ca2+]Lu). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released Ca2+ mainly from the basolateral ER-rich part of the cell. The rate of Ca2+ release from the ER was highly sensitive to the buffering of [Ca2+]c whereas ER Ca2+ refilling was enhanced by supplying free Ca2+ to the cytosol with [Ca2+]c clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM Ca2+. Elevation of extracellular Ca2+ to 10 mM from 1 mM raised resting [Ca2+]c slightly and often generated [Ca2+]c oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular Ca2+-sensing receptors linked to phospholipase C that mobilize Ca2+ from the ER, exposure of cells to 10 mM Ca2+ did not decrease [Ca2+]Lu but rather raised it. From these findings we conclude that 1) ER Ca2+ release is strictly regulated by feedback inhibition of [Ca2+]c, 2) ER Ca2+ refilling is determined by the rate of Ca2+ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular Ca2+-induced [Ca2+]c oscillations appear to be triggered not by activation of extracellular Ca2+-sensing receptors but by the ER sensitised by elevated [Ca2+]c and [Ca2+]Lu.     

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.     

The number and spatial distribution of IP3 receptors underlying calcium puffs in Xenopus oocytes

Calcium puffs are local Ca(2+) release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP(3)Rs) and serve as a basic "building block" from which global Ca(2+) waves are generated. Important questions remain as to the number of IP(3)Rs that open during a puff, their spatial distribution within a cluster, and how much Ca(2+) current flows through each channel. The recent discovery of "trigger" events-small Ca(2+) signals that immediately precede puffs and are interpreted to arise through opening of single IP(3)R channels-now provides a useful yardstick by which to calibrate the Ca(2+) flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca(2+) release in two sequential stages; representing the trigger by the opening of a single IP(3)R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP(3)Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca(2+) and Ca(2+)-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca(2+) buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25-35 IP(3)Rs, each carrying a Ca(2+) current of approximately 0.4 pA, with the channels distributed through a cluster 300-800 nm in diameter.     

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.     

Synchronous free Ca2+ changes in individual neutrophils stimulated by leukotriene B4.

Calcium (Ca2+) signals were monitored in individual neutrophils using ratio imaging of fura-2. In contrast to N-formyl-L-leucyl-L-phenylalanine (f-met-leu-phe), which produced grossly asynchronous Ca2+ signals with delays in response (up to 60 s), leukotriene B4 (LTB4) provoked synchronous and immediate elevations in cytosolic free Ca2+. Some individual neutrophils which responded immediately to LTB4, subsequently displayed delayed Ca2+ signals in response to f-met-leu-phe. A sub-population of neutrophils failed to respond to both LTB4 and f-met-leu-phe. The asynchrony of the Ca2+ signalling to f-met-leu-phe is not, therefore, an obligatory property of signal transduction in neutrophils.     

Mitochondrial organization and Ca2+ uptake

Mitochondria may function as multiple separate organelles or as a single electrically coupled continuum to modulate changes in [Ca2+]c (cytoplasmic Ca2+ concentration) in various cell types. Mitochondria may also be tethered to the internal Ca2+ store or plasma membrane in particular parts of cells to facilitate the organelles modulation of local and global [Ca2+]c increases. Differences in the organization and positioning contributes significantly to the at times apparently contradictory reports on the way mitochondria modulate [Ca2+]c signals. In the present paper, we review the organization of mitochondria and the organelles role in Ca2+ signalling.     

Different patterns of receptor-activated cytoplasmic Ca2+ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2+ buffering.

Agonist-specific cytosolic Ca2+ oscillation patterns can be observed in individual cells and these have been explained by the co-existence of separate oscillatory mechanisms. In pancreatic acinar cells activation of muscarinic receptors typically evokes sinusoidal oscillations whereas stimulation of cholecystokinin (CCK) receptors evokes transient oscillations consisting of Ca2+ waves with long intervals between them. We have monitored changes in the cytosolic Ca2+ concentration ([Ca2+]i) by measuring Ca2(+)-activated Cl- currents in single internally perfused mouse pancreatic acinar cells. With minimal intracellular Ca2+ buffering we found that low concentrations of both ACh (50 nM) and CCK (10 pM) evoked repetitive short-lasting Ca2+ spikes of the same duration and frequency, but the probability of a spike being followed by a longer and larger Ca2+ wave was low for ACh and high for CCK. The probability that the receptor-evoked shortlasting Ca2+ spikes would initiate more substantial Ca2+ waves was dramatically increased by intracellular perfusion with solutions containing high concentrations of the mobile low affinity Ca2+ buffers citrate (10-40 mM) or ATP (10-20 mM). The different Ca2+ oscillation patterns normally induced by ACh and CCK would therefore appear not to be caused by separate mechanisms. We propose that specific receptor-controlled modulation of Ca2+ signal spreading, either by regulation of Ca2+ uptake into organelles and/or cellular Ca2+ extrusion, or by changing the sensitivity of the Ca2(+)-induced Ca2+ release mechanism, can be mimicked experimentally by different degrees of cytosolic Ca2+ buffering and can account for the various cytosolic Ca2+ spike patterns.