Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells
1 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, PO Box 12233, Research Triangle Park, NC 27709, USA
Abstract
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.
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Introduction
In many cell types, Ca2+ signalling induced by neurotransmitters or hormones is initiated through cell membrane receptors coupled to phospholipase C (PLC) and the production of inositol 1,4,5-trisphosphate (IP3) (Berridge, 1993). Under conditions of maximal receptor activation, a biphasic Ca2+ signal is produced that is comprised of an initial Ca2+ release from the endoplasmic reticulum, followed by a sustained Ca2+ entry across the plasma membrane, which may result in a sustained plateau of [Ca2+]i above baseline. The Ca2+ entry that occurs under these conditions is believed, in most instances, to be activated as a result of depletion of intracellular Ca2+ stores and has been termed capacitative Ca2+ entry (CCE) (Putney, 1986; Berridge, 1995; Putney et al. 2001). However, recent studies have revealed other receptor-regulated mechanisms for activation of Ca2+ entry in certain cell types (Llopis et al. 1992; Byron & Taylor, 1995; Shuttleworth & Thompson, 1996b).
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In contrast to the sustained elevation of [Ca2+]i seen with high agonist concentrations, a more complex and repetitive cycling of [Ca2+]i, known as [Ca2+]i oscillations, commonly results from lower and, in some instances, physiological concentrations of agonists in many kinds of cells (Woods et al. 1986; Fewtrell, 1993; Thomas et al. 1996; Berridge, 1997; Shuttleworth, 1999). These oscillations depend upon complex mechanisms of regenerative intracellular signalling, either at the level of phospholipase C activity, or the action of IP3 on intracellular stores. The oscillations generally 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.
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There is strong evidence that PLC activation can regulate a number of different Ca2+-entry pathways, and there is controversy as to the precise role of Ca2+ entry, and as to the mode of Ca2+ entry associated with Ca2+ oscillations (Shuttleworth, 1999; Shuttleworth & Mignen, 2003). Some models suggest that CCE provides Ca2+ entry during oscillations (Berridge, 1992; Thomas et al. 1996), but a novel, non-capacitative calcium-entry mechanism has been proposed that involves agonist-activated generation of arachidonic acid (AA) and AA-induced Ca2+ entry (Shuttleworth, 1999).
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In this study, we have utilized pharmacological manoeuvres to demonstrate, first, that Ca2+ oscillations in human epithelial kidney (HEK293) cells are not obligatorily driven by Ca2+ entry, but rather depend upon entry for their maintenance. Second, we demonstrate that the entry associated with the oscillations appears to occur through the store-operated or capacitative calcium entry pathway.
Methods
Cell culture
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HEK293 cells, obtained from ATCC, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine and maintained in a humidified 95% air–5% CO2 incubator at 37°C. HEK293 cells stably expressing a green fluorescent peptide (GFP)-tagged TRPC3 (H-T3-G) were also maintained in culture as described by Trebak et al. (2003). In preparation for Ca2+ measurements, cells were cultured to about 70% confluence and then transferred onto 40 mm x 40 mm glass coverslips at two different cell densities (cells ml–1), low or high. Specifically, 0.5 ml of either a 400 000 cells ml–1 (high) or 60 000 cells ml–1 (low) cell suspension was transferred to the centre of the coverslip, and the cells allowed to attach for a period of 12 h. Additional DMEM was then added to the coverslip, and the cells maintained in culture for an additional 36 h before use in calcium measurements. Unless specified, cells were grown in the high density condition. Note that this condition did not result in a confluent layer of cells.
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Calcium measurements
Fluorescence measurements were made on HEK293 cells loaded with the calcium sensitive dye fura-5F. The use of the lower affinity (KD 400 nM) fura-5F greatly increased the proportion of cells which oscillated when compared with our earlier experience using fura-2 (Luo et al. 2001). Coverslips with attached cells were mounted in a Teflon chamber and incubated in DMEM with 1 μM of the acetoxymethy ester form of fura-5F (fura-5F AM, Molecular Probes, USA) for 25 min in a humidified 95% air–5% CO2 incubator at 37°C. Before [Ca2+]i measurements were made, cells were washed 3 times and incubated for 30 min at room temperature (25°C) in a Hepes-buffered salt solution (HBSS (mM): NaCl 120; KCl 5.4; Mg2Cl 0.8; Hepes 20; CaCl2 1.8 and glucose 10 mM, with pH 7.4 adjusted by NaOH). In these experiments, nominally Ca2+-free solutions were HBSS with no added CaCl2.
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Fluorescence was monitored by placing the Teflon chamber with fura-5F-loaded cells onto the stage of a Nikon TS-100 inverted microscope equipped with a 20 x fluor objective (0.75 NA). Fluorescence images of the cells were recorded and analysed with a digital fluorescence imaging system (InCyt Im2, Intracellular Imaging Inc., Cincinnati, OH, USA) equipped with a light-sensitive CCD camera (Cooke PixelFly, ASI, Eugene, OR, USA). Fura-5F fluorescence was monitored by alternately exciting the dye at 340 and 380 nm, and collecting the emission wavelength at 510 nm. Changes in intracellular calcium are represented as the ratio of fura-5F fluorescence due to excitation at 340 nm to that due to excitation at 380 nm (ratio = F340/F380). The ratio changes in fields of fura-5F-loaded cells were collected from a multiple regions of interest (ROI), with each ROI representing an individual cell. Typically, 25–35 ROIs were monitored per experiment. In all cases, ratio values have been corrected for contributions by autofluorescence, which is measured after treating cells with 10 μM ionomycin and 20 mM MnCl2. Peak ratio values typically were less than 2.0 while Rmax, determined in situ, averaged 6.5, indicating that the ratio values were less than half-saturation and thus essentially proportional to Ca2+ concentration.
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Analysis of agonist-induced calcium oscillations
In order to summarize the oscillatory Ca2+ responses to methacholine (MeCh) recorded from multiple ROIs in a given field of cells (see Fig. 1B and C for examples of 5 μM MeCh response), the following approach was taken. For each individual cell, the 25 min duration of the MeCh stimulation was separated into five successive 5 min time periods (the ‘0’ time point value represents a 5 min period prior to MeCh treatment). Within each 5 min time period, the number of Ca2+ spikes that occurred during that period was logged, as well as the peak ratio value for each Ca2+ spike. Subsequently, the number of Ca2+ spikes per 5 min time period was averaged for all responding cells in the field, as was the average ratio value for all observed calcium spikes for the same 5 min time period. These data are presented in Fig. 1D and E for 1 and 5 μM MeCh responses in HEK293 cells in the presence of 1.8 mM extracellular Ca2+. Generally the data for each condition are derived from 80 to 150 cells, taken from at least three independent experiments (different coverslips). The actual range of cell numbers are given in the figure legends.
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HEK293 cells loaded with fura-5F were treated with low concentrations of MeCh in HBSS containing 1.8 mM extracellular calcium. A, concentration–response relationship for MeCh in terms of the percentage of total cells responding (mean ± S.E.M., 0.1 μM, n = 3, 93 cells total; 0.5 μM, n = 3, 134 cells total; 1.0 μM, n = 7, 256 cells total; 5.0 μM, n = 9, 298 cells total). B, typical calcium response of a field of HEK293 cells to 5 μM MeCh. The 5 min data binning periods are indicated. C, examples of diverse 5 μM MeCh-induced calcium responses in single cells. D, effect of 1 μM () and 5 μM () MeCh (added at arrow) on oscillation frequency. E, effect of 1 μM () and 5 μM () MeCh (added at arrow, t = 0) on calcium spike magnitude. The data in D and E were extracted from the same experimental data performed at each MeCh concentration on the same day, and are means ± S.E.M. of 4 (for 1.0 μM) or 3 (for 5.0 μM) independent experiments, with a total of 69 (1.0 μM) or 62 (5.0 μM) cells.
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Materials
Methacholine was purchased from Sigma (St Louis, MO, USA), thapsigargin from Alexis (San Diego, CA, USA), fura-5F AM from Molecular Probes (Eugene, OR, USA) and 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) from CalBioChem (CA, USA). Arachidonic acid, 5,8,11,14-eicosatetraenoic acid, was obtained from BioMol (PA, USA). 2-Aminoethyoxydiphenyl borane (2-APB) synthesis was previously described (Broad et al. 2001).
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Results and Discussion
Characterization of [Ca2+]i response to MeCh in HEK293 cells loaded with the calcium sensitive dye fura-5F. A primary aim of this study was to characterize the oscillatory [Ca2+]i response in HEK293 cells to submaximal concentrations of the muscarinic receptor agonist MeCh. In a previous study, population measurements in HEK293 cells stimulated with MeCh demonstrated an apparent graded Ca2+ response, such that the degree of Ca2+ entry was proportional to the extent of Ca2+ pool depletion, and with an EC50 5 μM (Trebak et al. 2003).
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In Fig. 1A, we reassessed four concentrations from the lower range of the [MeCh] concentration–effect curve (0.1, 0.5, 1.0 and 5 μM), but by imaging a whole field of cells (typically 25–35 cells) and collecting data from the individual cells. We observed that with these low concentrations of MeCh at the single cell level, the individual responses varied considerably, ranging from a single [Ca2+]i spike to multiple spikes or, in a few instances, sustained [Ca2+]i responses or no response at all. Thus, we first assessed sensitivity to MeCh by plotting the percentage of cells responding to each [MeCh]. As shown in Fig. 1A, the percentage of cells responding to MeCh was concentration dependent, with a threshold between 0.1 and 0.5 μM, and with 95% of the cells responding to 5 μM MeCh. At concentrations above 5 μM, the percentage of cells oscillating decreases and responses are more sustained (data not shown).
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Figure 1B is a composite of multiple cells from one field of cells treated with 5 μM MeCh, recorded over a period of 25 min. There is considerable variety in the pattern of the Ca2+ oscillations recorded in individual cells, and Fig. 1C illustrates some of these responses. To summarize the composite data illustrated in Fig. 1B, two features were extracted as described in Methods, and presented in Fig. 1D and E. Specifically, the average frequency (Fig. 1D, Ca2+ spikes per 5 min period) and average peak calcium spike ratio (Fig. 1E: Peak ratio) were plotted for both a 1 μM and 5 μM MeCh response. As originally described for hepatocytes by Woods et al. (1986, 1987), the frequency of the calcium response is concentration dependent, whereas the magnitude of the calcium spikes is not.
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Both 1 μM and 5 μM MeCh appeared to generate a sustained oscillatory calcium response over the 25 min time period, but since 5 μM MeCh could induce sustained [Ca2+]i oscillations in 95% of HEK293 cells (compared with 60% with 1 μM MeCh), the 5 μM MeCh concentration was used for the remainder of the study.
Role of extracellular Ca2+ in MeCh-induced [Ca2+]i oscillations. In order to assess dependence of MeCh-induced [Ca2+]i oscillations on extracellular calcium, we compared the response of HEK293 cells to 5 μM MeCh in the presence of extracellular Ca2+ (1.8 mM) with that when extracellular Ca2+ was absent. The latter condition was obtained by either incubating the cells in nominally Ca2+-free HBSS, or nominally Ca2+-free HBSS supplemented with 200 μM of the Ca2+ chelator BAPTA. As demonstrated in Fig. 2A and summarized in Fig. 2B, reducing extracellular Ca2+ significantly reduces the sustained oscillating [Ca2+]i response to 5 μM MeCh. Removal of extracellular Ca2+ did not, however, significantly affect the percentage of responsive cells (control, 96.0 ± 2.0; – Ca2+, 86.4 ± 7.9; – Ca2+ + BAPTA, 89.0 ± 3.4), indicating that extracellular Ca2+ does not appear to influence receptor activation of phospholipase C.
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A, typical calcium response of a field of fura-5F-loaded HEK293 cells to 5 μM MeCh in the absence of extracellular calcium. The 5 min data binning periods are indicated. B, time course of 5 μM MeCh-induced calcium oscillation frequency in the presence (1.8 mM) or absence (nominally Ca2+-free with or without 200 μM BAPTA) of extracellular calcium. MeCh (5 μM) added at arrow. Means ± S.E.M. of: + Ca2+, 102 cells, 3 experiments; – Ca2+, 101 cells, 3 experiments; + BAPTA, 108 cells, 3 experiments.
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The data in Fig. 2 demonstrate the requirement for Ca2+ entry to maintain MeCh-induced [Ca2+]i oscillations, but they do not address the issue of whether or not the entry is a necessary initiating signal for the oscillations. To determine whether Ca2+ entry is necessary for triggering oscillations, we next monitored MeCh-induced [Ca2+]i oscillations under conditions where both the entry and efflux of Ca2+ ions across the plasma membrane are blocked. This condition effectively isolates, or insulates, the intracellular milieu from the calcium homeostatic role of the plasma membrane.
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To achieve this insulated condition, cells were incubated in the absence of extracellular calcium to preclude any means of calcium entry, and also in the presence of a high concentration of Gd3+ to block efflux of calcium via the plasma membrane Ca2+-ATPase. The use of high concentrations of lanthanides to block Ca2+ efflux from cells was first described by Van Breemen et al. (1972). The effectiveness of this ‘Gd3+-insulation’ condition is demonstrated in cells treated with thapsigargin in the absence of extracellular calcium (Fig. 3A). In the absence of extracellular calcium, the transient response of thapsigargin is a combination of the passive release of intracellularly stored calcium, followed by the efflux of this calcium across the plasma membrane via the plasma membrane Ca2+ pump. One millimolar [Gd3+] converts the transient thapsigargin response to one that is much more sustained (Fig. 3A), indicating that there is substantially reduced efflux of Ca2+ across the plasma membrane. The substantial increase in the [Ca2+]i signal indicates that the plasma membrane Ca2+-ATPase normally plays a significant role in buffering the release of Ca2+ to the cytoplasm under these conditions. Higher concentrations of Gd3+ produced [Ca2+]i responses that were even more sustained; however, above 1 mM, Gd3+ appeared to partially block muscarinic receptors resulting in a reduction in the number of cells showing oscillatory responses to 5 μM MeCh. Transient MeCh responses were observed with lower [Gd3+] that do not fully block calcium efflux. Treating cells with 5 μM MeCh in this ‘Gd3+-insulation’ condition gave a relatively sustained oscillatory [Ca2+]i response, similar to that seen in cells incubated in normal Ca2+-containing media (Fig. 3B, see also Fig. 5B). The shape (Fig. 4A) and magnitude (Fig. 4B) of the Ca2+ spikes were not significantly affected. Note that the effect of 1 mM Gd3+ is very different with this relatively low concentration of agonist than in the experiment with thapsigargin. With 5 μM MeCh, SERCA pumps continue to function, and apparently Ca2+ is fully capable of cycling intracellularly in an essentially normal manner. Thus the only effect of 1 mM Gd3+ in this case is preventing the small and gradual loss of Ca2+ via the plasma membrane Ca2+ pump, so that the oscillations can continue unabated in the absence of extracellular Ca2+. A similar result was recently reported by Sneyd et al. (2004). It is of interest that the magnitude of the [Ca2+]i spikes was not significantly affected by 1 mM Gd3+. This probably indicates very small contributions of both Ca2+ entry and Ca2+ pumping at the plasma membrane to individual spikes.
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A, effect of different concentrations of GdCl3 on the thapsigargin-induced calcium transient in HEK293 cells loaded with fura-5F and in the absence of extracellular calcium. All calcium traces are the average of 84–96 total cells from 3 independent experiments. B, effects of different concentrations of GdCl3 on the frequency of 5 μM MeCh-induced calcium oscillations in the absence of extracellular calcium. MeCh added at arrow (mean ± S.E.M. of 98–114 cells from 3 independent experiments).
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For all experiments in this figure, 1.8 mM extracellular Ca2+ was present throughout. A, effect of 1 μM GdCl3 on the sustained 5 μM MeCh-induced calcium oscillation frequency. HEK293 cells loaded with fura-5F were stimulated (at arrow) with 5 μM MeCh in the presence of extracellular calcium, and in the absence (control, ) and presence () of 1 μM GdCl3 (mean ± S.E.M. of 141–152 cells from 4 independent experiments). B, effect of 30 μM 2-APB on the 5 μM MeCh-induced calcium response. HEK293 cells loaded with fura-5F were stimulated (at arrow) with 5 μM MeCh in the presence of extracellular calcium, and in the absence (control, ) and presence () of 30 μM 2-APB. The effect of 2-APB on the MeCh-induced calcium response was also tested in the presence of 1 mM GdCl3 and in the absence of extracellular calcium (: Gd-insulation condition). All data are mean ± S.E.M. of 94–104 cells from 3 independent experiments. C, effect of depolarizing HEK293 cells with 65 mM K+ on the 5 μM MeCh-induced calcium response. The effect of 65 mM K+ on the MeCh-induced calcium response was also tested in the presence of 1 mM GdCl3 and in the absence of extracellular calcium (: Gd-insulation condition). All data are mean ± S.E.M. of 98–109 cells from 3 independent experiments. D, effect of 1 μM GdCl3 on percentage responsive cells compared with control from experiments in A. E, effect of 30 μM 2-APB on percentage responsive cells for experiments described in B. F, effect of 65 mM K+ on percentage responsive cells for experiments described in C.
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A, examples of traces showing Ca2+ spiking under control conditions (black trace), and in the absence of extracellular Ca2+ in the presence of 1 mM Gd3+ (grey trace). B, summary of spike amplitudes under the same conditions as in A. Means ± S.E.M. of 99 (control) and 76 (1 mM Gd3+) observations from 3 independent experiments.
These data indicate that the mechanisms underlying calcium oscillations are intrinsic to the intracellular milieu and that extracellular Ca2+ entry is not necessary to either initiate the MeCh-induced [Ca2+]i response or for the regenerative [Ca2+]i oscillation process.
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Pharmacological characterization of the Ca2+-entry process that maintains MeCh-induced [Ca2+]i oscillations
The data in Fig. 5 summarize the effects of Gd3+, 2-APB and membrane depolarization by elevated extracellular K+ on sustained MeCh-induced [Ca2+]i oscillations. While 2-APB is known to block other channel types, and Gd3+ will block other channel types at higher concentrations, there is not to our knowledge any mode of Ca2+ entry other than CCE which is blocked by 2-APB and low (μM) concentrations of Gd3+ (Putney, 2001). For example, Ca2+ entry activated by AA is insensitive to these reagents (Luo et al. 2001). Incubation of the cells with either 1 μM Gd3+ or 30 μM 2-APB inhibited the sustained oscillatory response to MeCh to an extent similar to that seen with removal of extracellular Ca2+ (Fig. 5A and B, respectively). This result therefore suggests that the Ca2+ entry that supports sustained Ca2+ oscillations is CCE.
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In a previous publication from this laboratory (Luo et al. 2001) we reported inhibition of [Ca2+]i oscillations in HEK293 cells by 2-APB, but not by Gd3+. The current findings indicate that the conclusion regarding the action of Gd3+ was incorrect. The difference apparently results from substantial technical improvement in the method of investigating oscillations in the current work. Specifically, the use of the lower affinity indicator results in almost all cells oscillating, while in the previous study, a minority of cells responded in this way. Additionally, the earlier study utilized photon counting permitting analysis of a single cell per experiment. In the current study, an imaging system with a low magnification objective was used, increasing the number of cells analysed by almost two orders of magnitude, and permitting statistical rather than the less reliable anecdotal analysis of the data.
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In addition to its effects as an inhibitor of store-operated channels, 2-APB can block IP3 receptors, at least in permeabilized cells (Maruyama et al. 1997). However, this does not seem to contribute to its action in blocking [Ca2+]i oscillations. The inhibitory effect of 2-APB was tested under Gd3+-insulation conditions with the reasoning that if 2-APB has any inhibitory effects upstream of calcium entry, or direct effects on [Ca2+]i release and oscillations, these effects should still be apparent. However, as shown in Fig. 5B, the MeCh-induced response was sustained in the presence of 2-APB and high [Gd3+], indicating that all of the inhibitory effects of 2-APB are on the Ca2+ entry component of the response. Consistent with this interpretation, 2-APB treatment, as well as 1 μM Gd3+, had no effect on the number of MeCh-responsive cells (Fig. 5D and E), indicating that the processes of receptor activation, PLC activation and IP3 action were not significantly affected by these agents.
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An alternative method of blocking or reducing calcium entry is to incubate the cells under depolarizing conditions, reducing the driving force for the entry of Ca2+ ions across the plasma membrane (for example see Shuttleworth & Thompson, 1996a, 1996b). In the current experiments, cells were stimulated with MeCh in HBSS modified to contain a depolarizing concentration of KCl (65 mM) (Bird et al. 2004). As expected, the elevated [K+] resulted in a depression of the sustained oscillations (Fig. 5C). However, we were surprised to observe that these depolarization conditions similarly inhibited the sustained oscillations under Gd3+-insulation conditions (Fig. 5C). This finding indicates that the inhibitory effect of elevated [K+] cannot be attributed simply to removing the driving force for calcium entry. Rather, this inhibitory effect must occur upstream of calcium entry and release, probably either affecting PLC activity, or receptor activation of PLC. Consistent with this conclusion is the observation that depolarizing cells reduces the percentage of MeCh-responsive cells to 65% (Fig. 5F).
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Effect of cell density on AA- and MeCh-induced calcium responses
Up to this point, all MeCh-induced calcium responses were tested on cells plated at high density as described in the Methods section. Unexpectedly, we found that AA (30 μM) (Luo et al. 2001) was ineffective at inducing a calcium response (Fig. 6A) (similar results were observed at higher [AA], data not shown). This observation is consistent with the finding that the entry pathway that supports [Ca2+]i oscillations is the store-operated one, not the AA-activated one. However, we considered that under different conditions, i.e. such that the cells were responsive to AA, an alternative mechanism might operate. We found that plating HEK293 cells at low density restored the AA-induced calcium response (Fig. 6A), and as previously shown by Luo et al. (2001) this response was comprised of both release of intracellular Ca2+ and Ca2+ entry across the plasma membrane (Fig. 6B). However, as shown in Fig. 6C, the sustained MeCh-induced [Ca2+]i oscillations in low density plated cells were also blocked by 1 μM Gd3+, suggesting the same role for CCE as in high density plated cells.
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A, as described in the Methods section, HEK293 cells were plated at low and high density. Fura-5F-loaded HEK293 cells were then treated with 30 μM AA in the presence of 1.8 mM extracellular calcium. The data presented are from a single experiment performed on the same day, with each trace the mean response from 30 cells. The data are representative of 5 similar experiments, covering a total of approximately 150 cells. B, the [Ca2+]i signal in low-density cells is comprised of Ca2+ release as well as Ca2+ entry across the plasma membrane. The protocol was as for the low-density cells in A, except that the medium contained no added Ca2+ initially, and was restored to 1.8 mM where indicated. The trace is a mean response from 13 cells, representative of 3 experiments with a total of approximately 45 cells. C, effect of 5 μM MeCh (added at arrow) on HEK293 cells grown at low cell density cell population. The control response to 5 μM MeCh () in the presence of 1.8 mM extracellular calcium was compared with the response in nominally Ca2+-free calcium conditions (), and that in the presence of 1 μM GdCl3 and 1.8 mM extracellular calcium (). Data are mean ± S.E.M. of 88–90 cells from 3 independent experiments.
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Why do MeCh-induced [Ca2+]i oscillations run down in the absence of calcium entry
The data thus far suggest that sustained MeCh-induced [Ca2+]i oscillations require the entry of extracellular calcium, mediated by CCE. In the absence of extracellular calcium entry [Ca2+]i oscillations run down and stop, the implication being that critical agonist-sensitive calcium stores are exhausted without calcium entry to replenish them. To address this, we compared the residual ionomycin-sensitive calcium stores in cells that had been treated for 25 min with MeCh in the presence or absence (e.g. Fig. 2B) of extracellular calcium. Under these conditions, ionomycin-sensitive stores are essentially coincident with the thapsigargin-sensitive stores (Ribeiro & Putney, 1996; Pedrosa Ribeiro et al. 2000); ionomycin is used because it produces a rapid release of Ca2+ such that the amplitude of the release transient is minimally affected by parallel Ca2+ buffering mechanisms. The expectation is that the ionomycin-sensitive calcium stores would be reduced when extracellular Ca2+ is not available to support the oscillations.
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As shown in Fig. 7, a substantial ionomycin-sensitive calcium store persists in cells treated with MeCh for 25 min whether in the presence or absence of Ca2+. The size of this Ca2+ store was slightly although significantly reduced in stimulated cells compared with unstimulated cells, and further slightly, but significantly reduced in cells stimulated in the absence of Ca2+. This suggests that run-down of the MeCh-induced [Ca2+]i oscillations in the absence of extracellular Ca2+ may involve exhaustion of a small, but critical, calcium pool. This is perhaps not surprising given the published evidence that only a small fraction of the endoplasmic reticulum is actually involved in communication with plasma membrane store-operated channels (Ribeiro & Putney, 1996; Parekh et al. 1997; Huang & Putney, 1998).
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A, following a 25 min incubation of fura-5F-loaded HEK293 cells under control conditions (continous line), or with 5 μM MeCh in the presence (dotted line) or absence (grey continuous line) of extracellular calcium, cells were treated with 20 μM ionomycin (+ 3 mM BAPTA) (data shown are mean responses from 26 to 28 cells in a single experiment, representative of 4 independent experiments). B, summarized data for the amplitude of the ionomycin-induced [Ca2+]i transient under the same conditions as in A. Means ± S.E.M. of 100–106 observations; *P < 0.001 compared with control; **P < 0.001 compared with MeCh + [Ca2+]o.
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Is CCE the only process of calcium entry that can sustain MeCh-induced [Ca2+]i oscillations
The data thus far indicate that CCE is the underlying physiological mechanism of calcium entry that supports MeCh-induced [Ca2+]i oscillations. But is CCE the only entry mechanism that can fulfil this function In addition, if there were additional pathways contributing to maintenance of oscillations, would this analysis detect them To test this, we used HEK293 cells stably expressing TRPC3 (H-T3-G cells), an agonist-activated calcium entry channel that is dependent on PLC activity but clearly not regulated by either IP3 or calcium pool depletion (Trebak et al. 2003). In both wild type HEK293 cells and H-T3-G cells, 5 μM MeCh induces a sustained [Ca2+]i oscillatory response (Fig. 8A and B, respectively). After monitoring the oscillations for 25 min, the cells were treated with 1 μM Gd3+ which will block CCE but not TRPC3 (Trebak et al. 2002). The effect of blocking CCE on the oscillation response was measured by comparing the frequency of [Ca2+]i oscillations after Gd3+ treatment (period b) as a percentage of the frequency before Gd3+ treatment (period a), and is summarized in Fig. 8C. As can be seen in Fig. 8B and C, the [Ca2+]i oscillations in H-T3-G cells are minimally affected by 1 μM Gd3+, as compared with wild type HEK293 cells. The approximate 30% drop in frequency in H-T3-G cells probably reflects the contribution made by CCE, but clearly the Gd3+-insensitive TRPC3 channels contribute to the Ca2+ entry and allow the oscillations to be sustained.
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Wild type HEK293 (A) or H-T3-G (B) cells were treated with 5 μM MeCh (as indicated) in the presence of 1.8 mM extracellular calcium. After 25 min, and at a point when calcium entry is critical for sustaining this response, the cells were treated with 1 μM GdCl3 to block CCE. C, the percentage of the control oscillation frequency after Gd3+ addition (frequency of b/frequency of a x 100%) for each cell type (mean ± S.E.M. of 92–93 cells from 3 independent experiments).
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These data suggest that the calcium entry process required to support agonist-induced [Ca2+]i oscillations need not necessarily be CCE. The sustained [Ca2+]i oscillatory response in TRPC3-expressing HEK293 cells clearly indicates agonist-activated non-capacitative calcium entry processes can support this process as well. However, in the case of the HEK293 cells used in this study, it is clear that CCE is the predominant, and probably only Ca2+-entry mechanism maintaining [Ca2+]i oscillations.
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Summary
The present study clearly demonstrates that CCE is the underlying mechanism of calcium entry that is required to sustain MeCh-induced [Ca2+]i oscillations in HEK293 cells. Interestingly, the observation that MeCh-induced [Ca2+]i oscillations can be sustained when transcellular calcium fluxes are prevented by Gd3+-insulation suggests that (i) the mechanisms underlying calcium oscillations are intrinsic to the intracellular milieu, (ii) Ca2+ entry is not necessary to initiate the MeCh-induced [Ca2+]i response, and (iii) calcium entry is not necessary for sustained [Ca2+]i oscillations if calcium ions are prevented from being pumped out of the cell. This latter point would suggest that the role for calcium entry is primarily to replenish any calcium ions that are lost from the cytoplasm and which may lead to exhaustion of a small, yet critical intracellular calcium pool. In addition, while CCE is the physiological mechanism of calcium entry supporting [Ca2+]i oscillations in HEK293 cells, the ability of TRPC3-mediated calcium entry to support this process alone suggests that the replenishment role of calcium entry can be supported by mechanisms other than CCE.
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Bird GJS, Wedel BJ, Lievremont J-P, Trebak M, Aziz O, Vazquez G & Putney JW Jr (2004). Mechanisms of phospholipase C-regulated calcium entry. Current Mol Med 4, 291–301.
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Abstract
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.
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Introduction
In many cell types, Ca2+ signalling induced by neurotransmitters or hormones is initiated through cell membrane receptors coupled to phospholipase C (PLC) and the production of inositol 1,4,5-trisphosphate (IP3) (Berridge, 1993). Under conditions of maximal receptor activation, a biphasic Ca2+ signal is produced that is comprised of an initial Ca2+ release from the endoplasmic reticulum, followed by a sustained Ca2+ entry across the plasma membrane, which may result in a sustained plateau of [Ca2+]i above baseline. The Ca2+ entry that occurs under these conditions is believed, in most instances, to be activated as a result of depletion of intracellular Ca2+ stores and has been termed capacitative Ca2+ entry (CCE) (Putney, 1986; Berridge, 1995; Putney et al. 2001). However, recent studies have revealed other receptor-regulated mechanisms for activation of Ca2+ entry in certain cell types (Llopis et al. 1992; Byron & Taylor, 1995; Shuttleworth & Thompson, 1996b).
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In contrast to the sustained elevation of [Ca2+]i seen with high agonist concentrations, a more complex and repetitive cycling of [Ca2+]i, known as [Ca2+]i oscillations, commonly results from lower and, in some instances, physiological concentrations of agonists in many kinds of cells (Woods et al. 1986; Fewtrell, 1993; Thomas et al. 1996; Berridge, 1997; Shuttleworth, 1999). These oscillations depend upon complex mechanisms of regenerative intracellular signalling, either at the level of phospholipase C activity, or the action of IP3 on intracellular stores. The oscillations generally 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.
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There is strong evidence that PLC activation can regulate a number of different Ca2+-entry pathways, and there is controversy as to the precise role of Ca2+ entry, and as to the mode of Ca2+ entry associated with Ca2+ oscillations (Shuttleworth, 1999; Shuttleworth & Mignen, 2003). Some models suggest that CCE provides Ca2+ entry during oscillations (Berridge, 1992; Thomas et al. 1996), but a novel, non-capacitative calcium-entry mechanism has been proposed that involves agonist-activated generation of arachidonic acid (AA) and AA-induced Ca2+ entry (Shuttleworth, 1999).
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In this study, we have utilized pharmacological manoeuvres to demonstrate, first, that Ca2+ oscillations in human epithelial kidney (HEK293) cells are not obligatorily driven by Ca2+ entry, but rather depend upon entry for their maintenance. Second, we demonstrate that the entry associated with the oscillations appears to occur through the store-operated or capacitative calcium entry pathway.
Methods
Cell culture
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HEK293 cells, obtained from ATCC, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine and maintained in a humidified 95% air–5% CO2 incubator at 37°C. HEK293 cells stably expressing a green fluorescent peptide (GFP)-tagged TRPC3 (H-T3-G) were also maintained in culture as described by Trebak et al. (2003). In preparation for Ca2+ measurements, cells were cultured to about 70% confluence and then transferred onto 40 mm x 40 mm glass coverslips at two different cell densities (cells ml–1), low or high. Specifically, 0.5 ml of either a 400 000 cells ml–1 (high) or 60 000 cells ml–1 (low) cell suspension was transferred to the centre of the coverslip, and the cells allowed to attach for a period of 12 h. Additional DMEM was then added to the coverslip, and the cells maintained in culture for an additional 36 h before use in calcium measurements. Unless specified, cells were grown in the high density condition. Note that this condition did not result in a confluent layer of cells.
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Calcium measurements
Fluorescence measurements were made on HEK293 cells loaded with the calcium sensitive dye fura-5F. The use of the lower affinity (KD 400 nM) fura-5F greatly increased the proportion of cells which oscillated when compared with our earlier experience using fura-2 (Luo et al. 2001). Coverslips with attached cells were mounted in a Teflon chamber and incubated in DMEM with 1 μM of the acetoxymethy ester form of fura-5F (fura-5F AM, Molecular Probes, USA) for 25 min in a humidified 95% air–5% CO2 incubator at 37°C. Before [Ca2+]i measurements were made, cells were washed 3 times and incubated for 30 min at room temperature (25°C) in a Hepes-buffered salt solution (HBSS (mM): NaCl 120; KCl 5.4; Mg2Cl 0.8; Hepes 20; CaCl2 1.8 and glucose 10 mM, with pH 7.4 adjusted by NaOH). In these experiments, nominally Ca2+-free solutions were HBSS with no added CaCl2.
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Fluorescence was monitored by placing the Teflon chamber with fura-5F-loaded cells onto the stage of a Nikon TS-100 inverted microscope equipped with a 20 x fluor objective (0.75 NA). Fluorescence images of the cells were recorded and analysed with a digital fluorescence imaging system (InCyt Im2, Intracellular Imaging Inc., Cincinnati, OH, USA) equipped with a light-sensitive CCD camera (Cooke PixelFly, ASI, Eugene, OR, USA). Fura-5F fluorescence was monitored by alternately exciting the dye at 340 and 380 nm, and collecting the emission wavelength at 510 nm. Changes in intracellular calcium are represented as the ratio of fura-5F fluorescence due to excitation at 340 nm to that due to excitation at 380 nm (ratio = F340/F380). The ratio changes in fields of fura-5F-loaded cells were collected from a multiple regions of interest (ROI), with each ROI representing an individual cell. Typically, 25–35 ROIs were monitored per experiment. In all cases, ratio values have been corrected for contributions by autofluorescence, which is measured after treating cells with 10 μM ionomycin and 20 mM MnCl2. Peak ratio values typically were less than 2.0 while Rmax, determined in situ, averaged 6.5, indicating that the ratio values were less than half-saturation and thus essentially proportional to Ca2+ concentration.
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Analysis of agonist-induced calcium oscillations
In order to summarize the oscillatory Ca2+ responses to methacholine (MeCh) recorded from multiple ROIs in a given field of cells (see Fig. 1B and C for examples of 5 μM MeCh response), the following approach was taken. For each individual cell, the 25 min duration of the MeCh stimulation was separated into five successive 5 min time periods (the ‘0’ time point value represents a 5 min period prior to MeCh treatment). Within each 5 min time period, the number of Ca2+ spikes that occurred during that period was logged, as well as the peak ratio value for each Ca2+ spike. Subsequently, the number of Ca2+ spikes per 5 min time period was averaged for all responding cells in the field, as was the average ratio value for all observed calcium spikes for the same 5 min time period. These data are presented in Fig. 1D and E for 1 and 5 μM MeCh responses in HEK293 cells in the presence of 1.8 mM extracellular Ca2+. Generally the data for each condition are derived from 80 to 150 cells, taken from at least three independent experiments (different coverslips). The actual range of cell numbers are given in the figure legends.
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HEK293 cells loaded with fura-5F were treated with low concentrations of MeCh in HBSS containing 1.8 mM extracellular calcium. A, concentration–response relationship for MeCh in terms of the percentage of total cells responding (mean ± S.E.M., 0.1 μM, n = 3, 93 cells total; 0.5 μM, n = 3, 134 cells total; 1.0 μM, n = 7, 256 cells total; 5.0 μM, n = 9, 298 cells total). B, typical calcium response of a field of HEK293 cells to 5 μM MeCh. The 5 min data binning periods are indicated. C, examples of diverse 5 μM MeCh-induced calcium responses in single cells. D, effect of 1 μM () and 5 μM () MeCh (added at arrow) on oscillation frequency. E, effect of 1 μM () and 5 μM () MeCh (added at arrow, t = 0) on calcium spike magnitude. The data in D and E were extracted from the same experimental data performed at each MeCh concentration on the same day, and are means ± S.E.M. of 4 (for 1.0 μM) or 3 (for 5.0 μM) independent experiments, with a total of 69 (1.0 μM) or 62 (5.0 μM) cells.
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Materials
Methacholine was purchased from Sigma (St Louis, MO, USA), thapsigargin from Alexis (San Diego, CA, USA), fura-5F AM from Molecular Probes (Eugene, OR, USA) and 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) from CalBioChem (CA, USA). Arachidonic acid, 5,8,11,14-eicosatetraenoic acid, was obtained from BioMol (PA, USA). 2-Aminoethyoxydiphenyl borane (2-APB) synthesis was previously described (Broad et al. 2001).
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Results and Discussion
Characterization of [Ca2+]i response to MeCh in HEK293 cells loaded with the calcium sensitive dye fura-5F. A primary aim of this study was to characterize the oscillatory [Ca2+]i response in HEK293 cells to submaximal concentrations of the muscarinic receptor agonist MeCh. In a previous study, population measurements in HEK293 cells stimulated with MeCh demonstrated an apparent graded Ca2+ response, such that the degree of Ca2+ entry was proportional to the extent of Ca2+ pool depletion, and with an EC50 5 μM (Trebak et al. 2003).
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In Fig. 1A, we reassessed four concentrations from the lower range of the [MeCh] concentration–effect curve (0.1, 0.5, 1.0 and 5 μM), but by imaging a whole field of cells (typically 25–35 cells) and collecting data from the individual cells. We observed that with these low concentrations of MeCh at the single cell level, the individual responses varied considerably, ranging from a single [Ca2+]i spike to multiple spikes or, in a few instances, sustained [Ca2+]i responses or no response at all. Thus, we first assessed sensitivity to MeCh by plotting the percentage of cells responding to each [MeCh]. As shown in Fig. 1A, the percentage of cells responding to MeCh was concentration dependent, with a threshold between 0.1 and 0.5 μM, and with 95% of the cells responding to 5 μM MeCh. At concentrations above 5 μM, the percentage of cells oscillating decreases and responses are more sustained (data not shown).
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Figure 1B is a composite of multiple cells from one field of cells treated with 5 μM MeCh, recorded over a period of 25 min. There is considerable variety in the pattern of the Ca2+ oscillations recorded in individual cells, and Fig. 1C illustrates some of these responses. To summarize the composite data illustrated in Fig. 1B, two features were extracted as described in Methods, and presented in Fig. 1D and E. Specifically, the average frequency (Fig. 1D, Ca2+ spikes per 5 min period) and average peak calcium spike ratio (Fig. 1E: Peak ratio) were plotted for both a 1 μM and 5 μM MeCh response. As originally described for hepatocytes by Woods et al. (1986, 1987), the frequency of the calcium response is concentration dependent, whereas the magnitude of the calcium spikes is not.
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Both 1 μM and 5 μM MeCh appeared to generate a sustained oscillatory calcium response over the 25 min time period, but since 5 μM MeCh could induce sustained [Ca2+]i oscillations in 95% of HEK293 cells (compared with 60% with 1 μM MeCh), the 5 μM MeCh concentration was used for the remainder of the study.
Role of extracellular Ca2+ in MeCh-induced [Ca2+]i oscillations. In order to assess dependence of MeCh-induced [Ca2+]i oscillations on extracellular calcium, we compared the response of HEK293 cells to 5 μM MeCh in the presence of extracellular Ca2+ (1.8 mM) with that when extracellular Ca2+ was absent. The latter condition was obtained by either incubating the cells in nominally Ca2+-free HBSS, or nominally Ca2+-free HBSS supplemented with 200 μM of the Ca2+ chelator BAPTA. As demonstrated in Fig. 2A and summarized in Fig. 2B, reducing extracellular Ca2+ significantly reduces the sustained oscillating [Ca2+]i response to 5 μM MeCh. Removal of extracellular Ca2+ did not, however, significantly affect the percentage of responsive cells (control, 96.0 ± 2.0; – Ca2+, 86.4 ± 7.9; – Ca2+ + BAPTA, 89.0 ± 3.4), indicating that extracellular Ca2+ does not appear to influence receptor activation of phospholipase C.
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A, typical calcium response of a field of fura-5F-loaded HEK293 cells to 5 μM MeCh in the absence of extracellular calcium. The 5 min data binning periods are indicated. B, time course of 5 μM MeCh-induced calcium oscillation frequency in the presence (1.8 mM) or absence (nominally Ca2+-free with or without 200 μM BAPTA) of extracellular calcium. MeCh (5 μM) added at arrow. Means ± S.E.M. of: + Ca2+, 102 cells, 3 experiments; – Ca2+, 101 cells, 3 experiments; + BAPTA, 108 cells, 3 experiments.
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The data in Fig. 2 demonstrate the requirement for Ca2+ entry to maintain MeCh-induced [Ca2+]i oscillations, but they do not address the issue of whether or not the entry is a necessary initiating signal for the oscillations. To determine whether Ca2+ entry is necessary for triggering oscillations, we next monitored MeCh-induced [Ca2+]i oscillations under conditions where both the entry and efflux of Ca2+ ions across the plasma membrane are blocked. This condition effectively isolates, or insulates, the intracellular milieu from the calcium homeostatic role of the plasma membrane.
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To achieve this insulated condition, cells were incubated in the absence of extracellular calcium to preclude any means of calcium entry, and also in the presence of a high concentration of Gd3+ to block efflux of calcium via the plasma membrane Ca2+-ATPase. The use of high concentrations of lanthanides to block Ca2+ efflux from cells was first described by Van Breemen et al. (1972). The effectiveness of this ‘Gd3+-insulation’ condition is demonstrated in cells treated with thapsigargin in the absence of extracellular calcium (Fig. 3A). In the absence of extracellular calcium, the transient response of thapsigargin is a combination of the passive release of intracellularly stored calcium, followed by the efflux of this calcium across the plasma membrane via the plasma membrane Ca2+ pump. One millimolar [Gd3+] converts the transient thapsigargin response to one that is much more sustained (Fig. 3A), indicating that there is substantially reduced efflux of Ca2+ across the plasma membrane. The substantial increase in the [Ca2+]i signal indicates that the plasma membrane Ca2+-ATPase normally plays a significant role in buffering the release of Ca2+ to the cytoplasm under these conditions. Higher concentrations of Gd3+ produced [Ca2+]i responses that were even more sustained; however, above 1 mM, Gd3+ appeared to partially block muscarinic receptors resulting in a reduction in the number of cells showing oscillatory responses to 5 μM MeCh. Transient MeCh responses were observed with lower [Gd3+] that do not fully block calcium efflux. Treating cells with 5 μM MeCh in this ‘Gd3+-insulation’ condition gave a relatively sustained oscillatory [Ca2+]i response, similar to that seen in cells incubated in normal Ca2+-containing media (Fig. 3B, see also Fig. 5B). The shape (Fig. 4A) and magnitude (Fig. 4B) of the Ca2+ spikes were not significantly affected. Note that the effect of 1 mM Gd3+ is very different with this relatively low concentration of agonist than in the experiment with thapsigargin. With 5 μM MeCh, SERCA pumps continue to function, and apparently Ca2+ is fully capable of cycling intracellularly in an essentially normal manner. Thus the only effect of 1 mM Gd3+ in this case is preventing the small and gradual loss of Ca2+ via the plasma membrane Ca2+ pump, so that the oscillations can continue unabated in the absence of extracellular Ca2+. A similar result was recently reported by Sneyd et al. (2004). It is of interest that the magnitude of the [Ca2+]i spikes was not significantly affected by 1 mM Gd3+. This probably indicates very small contributions of both Ca2+ entry and Ca2+ pumping at the plasma membrane to individual spikes.
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A, effect of different concentrations of GdCl3 on the thapsigargin-induced calcium transient in HEK293 cells loaded with fura-5F and in the absence of extracellular calcium. All calcium traces are the average of 84–96 total cells from 3 independent experiments. B, effects of different concentrations of GdCl3 on the frequency of 5 μM MeCh-induced calcium oscillations in the absence of extracellular calcium. MeCh added at arrow (mean ± S.E.M. of 98–114 cells from 3 independent experiments).
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For all experiments in this figure, 1.8 mM extracellular Ca2+ was present throughout. A, effect of 1 μM GdCl3 on the sustained 5 μM MeCh-induced calcium oscillation frequency. HEK293 cells loaded with fura-5F were stimulated (at arrow) with 5 μM MeCh in the presence of extracellular calcium, and in the absence (control, ) and presence () of 1 μM GdCl3 (mean ± S.E.M. of 141–152 cells from 4 independent experiments). B, effect of 30 μM 2-APB on the 5 μM MeCh-induced calcium response. HEK293 cells loaded with fura-5F were stimulated (at arrow) with 5 μM MeCh in the presence of extracellular calcium, and in the absence (control, ) and presence () of 30 μM 2-APB. The effect of 2-APB on the MeCh-induced calcium response was also tested in the presence of 1 mM GdCl3 and in the absence of extracellular calcium (: Gd-insulation condition). All data are mean ± S.E.M. of 94–104 cells from 3 independent experiments. C, effect of depolarizing HEK293 cells with 65 mM K+ on the 5 μM MeCh-induced calcium response. The effect of 65 mM K+ on the MeCh-induced calcium response was also tested in the presence of 1 mM GdCl3 and in the absence of extracellular calcium (: Gd-insulation condition). All data are mean ± S.E.M. of 98–109 cells from 3 independent experiments. D, effect of 1 μM GdCl3 on percentage responsive cells compared with control from experiments in A. E, effect of 30 μM 2-APB on percentage responsive cells for experiments described in B. F, effect of 65 mM K+ on percentage responsive cells for experiments described in C.
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A, examples of traces showing Ca2+ spiking under control conditions (black trace), and in the absence of extracellular Ca2+ in the presence of 1 mM Gd3+ (grey trace). B, summary of spike amplitudes under the same conditions as in A. Means ± S.E.M. of 99 (control) and 76 (1 mM Gd3+) observations from 3 independent experiments.
These data indicate that the mechanisms underlying calcium oscillations are intrinsic to the intracellular milieu and that extracellular Ca2+ entry is not necessary to either initiate the MeCh-induced [Ca2+]i response or for the regenerative [Ca2+]i oscillation process.
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Pharmacological characterization of the Ca2+-entry process that maintains MeCh-induced [Ca2+]i oscillations
The data in Fig. 5 summarize the effects of Gd3+, 2-APB and membrane depolarization by elevated extracellular K+ on sustained MeCh-induced [Ca2+]i oscillations. While 2-APB is known to block other channel types, and Gd3+ will block other channel types at higher concentrations, there is not to our knowledge any mode of Ca2+ entry other than CCE which is blocked by 2-APB and low (μM) concentrations of Gd3+ (Putney, 2001). For example, Ca2+ entry activated by AA is insensitive to these reagents (Luo et al. 2001). Incubation of the cells with either 1 μM Gd3+ or 30 μM 2-APB inhibited the sustained oscillatory response to MeCh to an extent similar to that seen with removal of extracellular Ca2+ (Fig. 5A and B, respectively). This result therefore suggests that the Ca2+ entry that supports sustained Ca2+ oscillations is CCE.
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In a previous publication from this laboratory (Luo et al. 2001) we reported inhibition of [Ca2+]i oscillations in HEK293 cells by 2-APB, but not by Gd3+. The current findings indicate that the conclusion regarding the action of Gd3+ was incorrect. The difference apparently results from substantial technical improvement in the method of investigating oscillations in the current work. Specifically, the use of the lower affinity indicator results in almost all cells oscillating, while in the previous study, a minority of cells responded in this way. Additionally, the earlier study utilized photon counting permitting analysis of a single cell per experiment. In the current study, an imaging system with a low magnification objective was used, increasing the number of cells analysed by almost two orders of magnitude, and permitting statistical rather than the less reliable anecdotal analysis of the data.
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In addition to its effects as an inhibitor of store-operated channels, 2-APB can block IP3 receptors, at least in permeabilized cells (Maruyama et al. 1997). However, this does not seem to contribute to its action in blocking [Ca2+]i oscillations. The inhibitory effect of 2-APB was tested under Gd3+-insulation conditions with the reasoning that if 2-APB has any inhibitory effects upstream of calcium entry, or direct effects on [Ca2+]i release and oscillations, these effects should still be apparent. However, as shown in Fig. 5B, the MeCh-induced response was sustained in the presence of 2-APB and high [Gd3+], indicating that all of the inhibitory effects of 2-APB are on the Ca2+ entry component of the response. Consistent with this interpretation, 2-APB treatment, as well as 1 μM Gd3+, had no effect on the number of MeCh-responsive cells (Fig. 5D and E), indicating that the processes of receptor activation, PLC activation and IP3 action were not significantly affected by these agents.
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An alternative method of blocking or reducing calcium entry is to incubate the cells under depolarizing conditions, reducing the driving force for the entry of Ca2+ ions across the plasma membrane (for example see Shuttleworth & Thompson, 1996a, 1996b). In the current experiments, cells were stimulated with MeCh in HBSS modified to contain a depolarizing concentration of KCl (65 mM) (Bird et al. 2004). As expected, the elevated [K+] resulted in a depression of the sustained oscillations (Fig. 5C). However, we were surprised to observe that these depolarization conditions similarly inhibited the sustained oscillations under Gd3+-insulation conditions (Fig. 5C). This finding indicates that the inhibitory effect of elevated [K+] cannot be attributed simply to removing the driving force for calcium entry. Rather, this inhibitory effect must occur upstream of calcium entry and release, probably either affecting PLC activity, or receptor activation of PLC. Consistent with this conclusion is the observation that depolarizing cells reduces the percentage of MeCh-responsive cells to 65% (Fig. 5F).
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Effect of cell density on AA- and MeCh-induced calcium responses
Up to this point, all MeCh-induced calcium responses were tested on cells plated at high density as described in the Methods section. Unexpectedly, we found that AA (30 μM) (Luo et al. 2001) was ineffective at inducing a calcium response (Fig. 6A) (similar results were observed at higher [AA], data not shown). This observation is consistent with the finding that the entry pathway that supports [Ca2+]i oscillations is the store-operated one, not the AA-activated one. However, we considered that under different conditions, i.e. such that the cells were responsive to AA, an alternative mechanism might operate. We found that plating HEK293 cells at low density restored the AA-induced calcium response (Fig. 6A), and as previously shown by Luo et al. (2001) this response was comprised of both release of intracellular Ca2+ and Ca2+ entry across the plasma membrane (Fig. 6B). However, as shown in Fig. 6C, the sustained MeCh-induced [Ca2+]i oscillations in low density plated cells were also blocked by 1 μM Gd3+, suggesting the same role for CCE as in high density plated cells.
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A, as described in the Methods section, HEK293 cells were plated at low and high density. Fura-5F-loaded HEK293 cells were then treated with 30 μM AA in the presence of 1.8 mM extracellular calcium. The data presented are from a single experiment performed on the same day, with each trace the mean response from 30 cells. The data are representative of 5 similar experiments, covering a total of approximately 150 cells. B, the [Ca2+]i signal in low-density cells is comprised of Ca2+ release as well as Ca2+ entry across the plasma membrane. The protocol was as for the low-density cells in A, except that the medium contained no added Ca2+ initially, and was restored to 1.8 mM where indicated. The trace is a mean response from 13 cells, representative of 3 experiments with a total of approximately 45 cells. C, effect of 5 μM MeCh (added at arrow) on HEK293 cells grown at low cell density cell population. The control response to 5 μM MeCh () in the presence of 1.8 mM extracellular calcium was compared with the response in nominally Ca2+-free calcium conditions (), and that in the presence of 1 μM GdCl3 and 1.8 mM extracellular calcium (). Data are mean ± S.E.M. of 88–90 cells from 3 independent experiments.
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Why do MeCh-induced [Ca2+]i oscillations run down in the absence of calcium entry
The data thus far suggest that sustained MeCh-induced [Ca2+]i oscillations require the entry of extracellular calcium, mediated by CCE. In the absence of extracellular calcium entry [Ca2+]i oscillations run down and stop, the implication being that critical agonist-sensitive calcium stores are exhausted without calcium entry to replenish them. To address this, we compared the residual ionomycin-sensitive calcium stores in cells that had been treated for 25 min with MeCh in the presence or absence (e.g. Fig. 2B) of extracellular calcium. Under these conditions, ionomycin-sensitive stores are essentially coincident with the thapsigargin-sensitive stores (Ribeiro & Putney, 1996; Pedrosa Ribeiro et al. 2000); ionomycin is used because it produces a rapid release of Ca2+ such that the amplitude of the release transient is minimally affected by parallel Ca2+ buffering mechanisms. The expectation is that the ionomycin-sensitive calcium stores would be reduced when extracellular Ca2+ is not available to support the oscillations.
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As shown in Fig. 7, a substantial ionomycin-sensitive calcium store persists in cells treated with MeCh for 25 min whether in the presence or absence of Ca2+. The size of this Ca2+ store was slightly although significantly reduced in stimulated cells compared with unstimulated cells, and further slightly, but significantly reduced in cells stimulated in the absence of Ca2+. This suggests that run-down of the MeCh-induced [Ca2+]i oscillations in the absence of extracellular Ca2+ may involve exhaustion of a small, but critical, calcium pool. This is perhaps not surprising given the published evidence that only a small fraction of the endoplasmic reticulum is actually involved in communication with plasma membrane store-operated channels (Ribeiro & Putney, 1996; Parekh et al. 1997; Huang & Putney, 1998).
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A, following a 25 min incubation of fura-5F-loaded HEK293 cells under control conditions (continous line), or with 5 μM MeCh in the presence (dotted line) or absence (grey continuous line) of extracellular calcium, cells were treated with 20 μM ionomycin (+ 3 mM BAPTA) (data shown are mean responses from 26 to 28 cells in a single experiment, representative of 4 independent experiments). B, summarized data for the amplitude of the ionomycin-induced [Ca2+]i transient under the same conditions as in A. Means ± S.E.M. of 100–106 observations; *P < 0.001 compared with control; **P < 0.001 compared with MeCh + [Ca2+]o.
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Is CCE the only process of calcium entry that can sustain MeCh-induced [Ca2+]i oscillations
The data thus far indicate that CCE is the underlying physiological mechanism of calcium entry that supports MeCh-induced [Ca2+]i oscillations. But is CCE the only entry mechanism that can fulfil this function In addition, if there were additional pathways contributing to maintenance of oscillations, would this analysis detect them To test this, we used HEK293 cells stably expressing TRPC3 (H-T3-G cells), an agonist-activated calcium entry channel that is dependent on PLC activity but clearly not regulated by either IP3 or calcium pool depletion (Trebak et al. 2003). In both wild type HEK293 cells and H-T3-G cells, 5 μM MeCh induces a sustained [Ca2+]i oscillatory response (Fig. 8A and B, respectively). After monitoring the oscillations for 25 min, the cells were treated with 1 μM Gd3+ which will block CCE but not TRPC3 (Trebak et al. 2002). The effect of blocking CCE on the oscillation response was measured by comparing the frequency of [Ca2+]i oscillations after Gd3+ treatment (period b) as a percentage of the frequency before Gd3+ treatment (period a), and is summarized in Fig. 8C. As can be seen in Fig. 8B and C, the [Ca2+]i oscillations in H-T3-G cells are minimally affected by 1 μM Gd3+, as compared with wild type HEK293 cells. The approximate 30% drop in frequency in H-T3-G cells probably reflects the contribution made by CCE, but clearly the Gd3+-insensitive TRPC3 channels contribute to the Ca2+ entry and allow the oscillations to be sustained.
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Wild type HEK293 (A) or H-T3-G (B) cells were treated with 5 μM MeCh (as indicated) in the presence of 1.8 mM extracellular calcium. After 25 min, and at a point when calcium entry is critical for sustaining this response, the cells were treated with 1 μM GdCl3 to block CCE. C, the percentage of the control oscillation frequency after Gd3+ addition (frequency of b/frequency of a x 100%) for each cell type (mean ± S.E.M. of 92–93 cells from 3 independent experiments).
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These data suggest that the calcium entry process required to support agonist-induced [Ca2+]i oscillations need not necessarily be CCE. The sustained [Ca2+]i oscillatory response in TRPC3-expressing HEK293 cells clearly indicates agonist-activated non-capacitative calcium entry processes can support this process as well. However, in the case of the HEK293 cells used in this study, it is clear that CCE is the predominant, and probably only Ca2+-entry mechanism maintaining [Ca2+]i oscillations.
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Summary
The present study clearly demonstrates that CCE is the underlying mechanism of calcium entry that is required to sustain MeCh-induced [Ca2+]i oscillations in HEK293 cells. Interestingly, the observation that MeCh-induced [Ca2+]i oscillations can be sustained when transcellular calcium fluxes are prevented by Gd3+-insulation suggests that (i) the mechanisms underlying calcium oscillations are intrinsic to the intracellular milieu, (ii) Ca2+ entry is not necessary to initiate the MeCh-induced [Ca2+]i response, and (iii) calcium entry is not necessary for sustained [Ca2+]i oscillations if calcium ions are prevented from being pumped out of the cell. This latter point would suggest that the role for calcium entry is primarily to replenish any calcium ions that are lost from the cytoplasm and which may lead to exhaustion of a small, yet critical intracellular calcium pool. In addition, while CCE is the physiological mechanism of calcium entry supporting [Ca2+]i oscillations in HEK293 cells, the ability of TRPC3-mediated calcium entry to support this process alone suggests that the replenishment role of calcium entry can be supported by mechanisms other than CCE.
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