Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.     

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.     

Thapsigargin reveals evidence for fMLP-insensitive calcium pools in human leukocytes.

To investigate the relationship between different intracellular Ca2+ pools, cytosolic free calcium ([Ca2+]i) was surveyed by means of a Fura-2 fluorescence ratio method on single isolated human leukocytes. Both monocytes and neutrophilic granulocytes (PMN) displayed long lasting spontaneous [Ca2+]i transient changes (1-2 min). In PMN stimulated with the bacterial peptide fMLP we observed transients with shorter duration (10-30 s) and smaller amplitude often superimposed on the long lasting transients. The time course of changes in [Ca2+]i was recorded in a large number (149) of single leukocytes prestimulated for 5 min with fMLP and then challenged with thapsigargin (a blocker of Ca2+ uptake in intracellular pools). Statistical analysis of [Ca2+]i responses revealed that fMLP-sensitive pools contributed to the long lasting [Ca2+]i transients seen in both leukocyte types. However, the existence of fMLP-insensitive calcium pools may explain the superimposed transients seen in PMN. Thapsigargin was also added together with EGTA (to impede contribution from extracellular Ca2+) to 198 fMLP prestimulated and 153 unstimulated PMN. Based on Ca2+ registrations in these cells and a mathematical model (supposing two separate first order responses) the amount of Ca2+ stored in the various pools and their release kinetics were estimated. The results indicate that fMLP-insensitive calcium pools exist in PMN but not in monocytes. Since the digital imaging technique also depicts cellular motility, an additional finding was that the leukocyte's ability to sequestrate the Ca2+ from the cytosol seemed important to locomotion.     

Calcium dynamics associated with a single action potential in a CNS presynaptic terminal.

Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the basis of a single compartment model. A single action potential led to a total Ca2+ influx of 0.8-1 pC. The accessible volume of the terminal was about 0.4 pl; thus the total calcium concentration increased by 10-13 microM. The Ca(2+)-binding ratio of the endogenous buffer was about 40, as estimated from the competition with Fura-2, indicating that 2.5% of the total calcium remained free. This is consistent with the peak increase in free calcium concentration of about 400 nM, which was measured directly with MagFura-2. The decay of the [Ca2+]i transients was fast, with time constants of 100 ms at 23 degrees C and 45 ms at 35 degrees C, indicating Ca2+ extrusion rates of 400 and 900 s-1, respectively. The combination of the relatively low endogenous Ca(2+)-binding ratio and the high rate of Ca2+ extrusion provides an efficient mechanism for rapidly removing the large Ca2+ load of the terminal evoked by an action potential.     

Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts

Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.     

Dynamic and static calcium gradients inside large snail (Helix aspersa) neurones detected with calcium-sensitive microelectrodes

We have used quartz Ca2+-sensitive microelectrodes (CASMs) in large voltage-clamped snail neurones to investigate the inward spread of Ca2+ after a brief depolarisation. Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 microm of the far side of the cell the [Ca2+]i transient time to peak was 4.4+/-0.5s, rising to 14.7+/-0.7s at a distance of 80 microm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 microm2 s(-1) and decreased in size by half over a distance of about 45 microm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90+/-20 microm2 s(-1).This may assist fast recovery by extrusion.     

Interferon-gamma elicits a G-protein-dependent Ca2+ signal in human neutrophils after depletion of intracellular Ca2+ stores

Interferon-gamma (IFN-gamma) has multiple effects on Ca2+ signalling in polymorphonuclear neutrophils (PMNs), including evoked cytosolic Ca2+ transients, increased capacitative calcium influx and increased sequestration of Ca2+ in intracellular stores. The present study was conducted to elucidate the mechanism behind the Ca2+ transients. As observed before, the IFN-gamma-evoked Ca2+ signals were apparent when extracellular Ca2+ was removed. A new finding was that the proportion of responding cells and the extent of calcium release increased with increasing time in EGTA buffer. As assessed by N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated Ca2+ release, the intracellular stores were depleted during this incubation period, and the extent of depletion correlated well with the appearance of IFN-gamma-induced Ca2+ signals. This store dependence of the IFN-gamma-induced Ca2+ signals was confirmed by the appearance of IFN-gamma-evoked Ca2+ signals in the presence of extracellular Ca2+ after store depletion by thapsigargin. The appearance of IFN-gamma-mediated Ca2+-signals in the presence of EGTA indicates that IFN-gamma stimulates Ca2+ release from intracellular stores. This was confirmed by the inability of the calcium transportation blocker La3+ to abolish the IFN-gamma response and the total abrogation of the response by the phospholipase C inhibitor U73122. Although these latter results imply a role for inositol 1,4,5-trisphosphate(IP3) in IFN-gamma signalling, comparison of IFN-gamma-evoked responses with fMLP responses revealed clear differences that suggest different signal-transduction pathways. However, responses to fMLP and IFN-gamma were both depressed by pertussis toxin, and the IFN-gamma responses were, in addition, inhibited by the tyrosine kinase inhibitor genistein. Further evidence of the involvement of tyrosine kinase was a slight stimulatory effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate. The PI-3K activity was of minor importance. In conclusion, we present evidence of a novel signal-transduction mechanism for IFN-gamma in PMNs, dependent on tyrosine kinase activity, a pertussis toxin-sensitive G protein and phospholipase C activity.     

Selective, high-resolution fluorescence imaging of mitochondrial Ca2+ concentration.

We have developed a digital image processing technique based on highpass filtering of microfluorimetric images for selective transmission of fine image details corresponding to mitochondria. This technique enabled the detection of the mitochondrial calcium signals with high selectivity, simultaneously with the cytosolic calcium signal. The validity of this technique was supported in primary cultures of rat brain capillary endothelial cells loaded with X-rhod-1 by the results that (i) inhibition of the mitochondrial Ca2+ uptake by discharging the mitochondrial membrane potential selectively abolished the transient of the highpass filtered signal evoked by ATP, and (ii) CGP-37157, a selective blocker of the mitochondrial Na+/Ca2+ exchanger, increased the peak amplitude of highpass filtered (mitochondrial) Ca2+ transients and caused a sustained plateau. The highpass filtering technique enabled the analysis of the mitochondrial Ca2+ transients in high temporal resolution. We found a uniform and monophasic rise of [Ca2+] in the mitochondrial population of the cell, following the cytosolic [Ca2+] with a delay at onset and peak. The introduced highpass filtering technique is a powerful tool in the high spatial and temporal resolution analysis of mitochondrial calcium transients, and it could be especially important in specimens where genetically targeted probes fail.     

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Calcium transients in cerebellar granule cell presynaptic terminals.

Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons.     

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca2+]i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca2+]i. We observed small, brief, Ca2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.     

A theoretical study of calcium microdomains in turtle hair cells.

Confocal imaging has revealed microdomains of intracellular free Ca2+ in turtle hair cells evoked by depolarizing pulses and has delineated factors affecting the growth and dissipation of such domains. However, imaging experiments have limited spatial and temporal resolution. To extend the range of the results we have developed a three-dimensional model of Ca2+ diffusion in a cylindrical hair cell, allowing part of the Ca2+ influx to occur over a small circular region (radius 0.125-1.0 micron) representing a high-density array of voltage-dependent channels. The model incorporated experimental information about the number of channels, the fixed and mobile Ca2+ buffers, and the Ca2+ extrusion mechanism. A feature of the calculations was the use of a variable grid size depending on the proximity to the Ca2+ channel cluster. The results agreed qualitatively with experimental data on the localization of the Ca2+ transients, although the experimental responses were smaller and slower, which is most likely due to temporal and spatial averaging in the imaging. The model made predictions about 1) the optimal Ca2+ channel number and density within a cluster, 2) the conditions to ensure independence of neighboring clusters, and 3) the influence of the Ca2+ buffers on the kinetics and localization of the microdomains. We suggest that an increase in the mobile Ca2+ buffer concentration in high-frequency hair cells (which possess a larger number of release sites) would allow lower amplitude and faster Ca2+ responses and promote functional independence of the sites.     

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.     

Increases in cytosolic calcium ion concentration can be dissociated from the killing of cultured hepatocytes by tert-butyl hydroperoxide

Digital imaging fluorescence microscopy was used to study the effect of tert-butyl hydroperoxide (TBHP) on the cytosolic free calcium concentration ([Ca2+]i) of single rat hepatocytes in primary culture. Within minutes of the addition of TBHP, individual hepatocytes displayed one or more peaks of increased [Ca2+]i that promptly returned to the prestimulation level. This was followed by a slower increase of [Ca2+]i that reached a plateau of 696 +/- 260 nM (basal 194 +/- nM) after 20 min. Another rise in [Ca2+]i, abrupt and much larger, preceded the death of the cells after about 45 min. Pretreatment of the hepatocytes with deferoxamine, a ferric iron chelator, or the addition of the antioxidants N,N'-diphenyl-p-phenylenediamine or catechol prevented the loss of viability. Neither the number of hepatocytes displaying the initial [Ca2+]i transients nor the magnitude of these oscillations was affected by deferoxamine, N,N'-diphenyl-p-phenyl-enediamine, or catechol. However, both the plateau phase and the abrupt rise in [Ca2+]i were prevented. Treatment of the hepatocytes with TBHP in a low calcium buffer (less than 2 microM Ca2+) reduced or abolished the initial [Ca2+]i transients and eliminated both the plateau phase and abrupt rise in [Ca2+]i. The onset of cell death was delayed by 10 min in the low calcium medium. Addition of 3.5 mM EGTA to the cultures lowered the basal calcium concentration, prevented both the initial [Ca2+]i spikes and the delayed changes, and further prolonged the onset of cell death. These data indicate that the killing of the cultured hepatocytes by TBHP can be dissociated from changes in intracellular calcium homeostasis. An influx of extracellular Ca2+ ions may aggravate somewhat the mechanisms of cell injury by an oxidative stress and accelerate the time of onset of cell death.     

Calcium signal compartmentalization

Cytosolic calcium signals are produced by suddenly increasing the concentration of free calcium ions (Ca2+). This can occur by opening channels permeable to Ca2+ either in the surface cell membrane or in the membranes of intracellular organelles containing high Ca2+ concentrations. Ca2+ signals can control several different processes, even in the same cell. In pancreatic acinar cells, for example, Ca2+ signals do not only control the normal secretion of digestive enzymes, but can also activate autodigestion and programmed cell death. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited. The mechanisms responsible for Ca2+ signal compartmentalization are now largely known and will be described on the basis of recent studies of Ca2+ transport pathways and their regulation in pancreatic acinar cells. It turns out that the Ca2+ handling as well as the structural characteristics of the endoplasmic reticulum (ER) and the mitochondria are of particular importance. Using a variety of Ca(2+)-sensitive fluorescent probes placed in different sub-cellular compartments in combination with local uncaging of caged Ca2+, many new insights into Ca2+ signal generation, compartmentalization and termination have recently been obtained.     

Quantitation of lymphocyte intracellular free calcium signals using indo-1

The calcium-responsive fluorescent dye indo-1 has been used in lymphocyte suspensions to measure changes in internal free calcium concentration, [Ca2+]i, in response to crosslinking of cell surface immunoglobulin. The quantitation of [Ca2+]i requires that indo-am ester used to load the cells be completely hydrolyzed to the indo-1 form inside the cells. This is shown to be greatly facilitated in the lymphocyte by the detergent Pluronic F-127. The quantitation of [Ca2+]i transients also requires an estimate of the fraction of the cells which contribute to the observed changes. The use of excessive amounts of intracellular dye can buffer [Ca2+]i transients and this effect has been used to estimate the size of the pool of calcium which is available for release when the B cell is stimulated by anti-immunoglobulin.     

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.     

Advancing age alters rapid and spontaneous refilling of caffeine-sensitive calcium stores in sympathetic superior cervical ganglion cells.

Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature.     

Imaging single-channel calcium microdomains

The Ca(2+) microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca(2+) signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca(2+) imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of 'optical patch-clamp recording' is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca(2+) ions.