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The Journal of Immunology, 2007, 179, 5255 -5263
Copyright © 2007 by The American Association of Immunologists, Inc.
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All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells1

Wei-Chiao Chang, Joseph Di Capite, Charmaine Nelson and Anant B. Parekh2

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 Disclosures
 References
 
In nonexcitable cells, receptor stimulation evokes Ca2+ release from the endoplasmic reticulum stores followed by Ca2+ influx through store-operated Ca2+ channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca2+ release-activated Ca2+ (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca2+-dependent activation of protein kinase C{alpha} and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca2+-dependent phospholipase A2 and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C4. In cell population studies, ERK activation, arachidonic acid release, and leukotriene C4 secretion were all graded with stimulus intensity. However, at a single cell level, Ca2+ influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca2+ influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca2+entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgement
 Disclosures
 References
 
In eukaryotic cells, Ca2+ influx is a critical trigger for a diverse array of cellular responses including exocytosis, muscle contraction, gene transcription, and cell growth (1). In nonexcitable cells, Ca2+ entry occurs through store-operated and second messenger-gated Ca2+ channels in the plasma membrane (2, 3). Store-operated Ca2+ channels are activated following the emptying of intracellular Ca2+ stores, are regulated by a diverse range of agonists, and are found in a variety of nonexcitable cells.

Electrophysiological evidence indicates that store-operated channels represent a heterogeneous family (4). The best characterized member is the Ca2+ release-activated Ca2+ (CRAC)3 channel and the whole cell current flowing through CRAC channels is called ICRAC (5, 6). Defective CRAC channel activity has been causally linked to inherited primary immunodeficiencies (7) and Ca2+ entry through CRAC channels regulates exocytosis (8), gene transcription (9), and cytoplasmic enzymes such as NO synthase (10), adenylate cyclase (11), and Ca2+-dependent phospholipase A2 (cPLA2; Ref. 12). Ca2+ influx through CRAC channels activates cPLA2 indirectly, via recruitment of conventional protein kinase C{alpha} and betaI isozymes and subsequent stimulation of the mitogen activated protein kinases ERK1 and ERK2 (13). Arachidonic acid liberated by cPLA2 is then converted to the proinflammatory paracrine signal leukotriene C4 (LTC4) by the 5-lipoxygenase enzyme (12, 13).

The physiological trigger for CRAC channel activation is an increase in the levels of the ubiquitous second messenger inositol 1,4,5-trisphosphate (InsP3), which occurs following stimulation of cell surface receptors that couple to phospholipase C (2, 14). The extent of ICRAC activation is steeply related to the cytoplasmic InsP3 concentration with a Hill coefficient of 12, and this essentially all-or-none relationship is seen either by infusing cells with InsP3 or following stimulation of plasma membrane receptors that activate phospholipase C (15). Such nonlinear behavior arises from the metabolism of InsP3 by inositol 5-phosphatase because the nonmetabolisable InsP3 analog Ins2,4,5P3 generates a graded response whereas InsP3-F, another analog that is only broken down by the 5-phosphatase, activates ICRAC in a highly supralinear manner (15, 16). Consistent with this is the finding that inhibition of inositol 5-phosphatase with InsP4 reduces the steepness of the relationship between InsP3 concentration and activation of ICRAC and enables lower concentrations of InsP3 to evoke robust Ca2+ entry (17). Hence, following stimulation of receptors that elevate intracellular InsP3 levels, ICRAC appears to activate in an essentially all-or-none manner. Although several negative feedback mechanisms can subsequently inhibit ICRAC and hence grade the overall extent of Ca2+ influx (2, 18), the fact that ICRAC activates in an all-or-none manner following receptor stimulation nevertheless raises an intriguing paradox. Weak stimuli (presented as concentrations of agonist that are below the affinity of the receptor for its cognate ligand) often produce weak cellular responses whereas higher concentrations elicit stronger responses. If ICRAC is essentially an all-or-none process, how can responses be graded with stimulus intensity?

We have addressed this issue by examining the effect of different levels of receptor activation on ICRAC, Ca2+ entry, protein kinase C{alpha}, and ERK stimulation and subsequent activation of the cPLA2 and 5-lipoxygenase enzymes in a mast cell line as well as in acutely isolated rat peritoneal mast cells. Our new results demonstrate that increasing agonist concentration increases protein kinase C{alpha} and ERK activation, cPLA2 stimulation, and LTC4 secretion in a graded manner. However, activation of ICRAC and Ca2+ influx remain essentially all-or-none processes. Responses appear graded with stimulus intensity because increasing agonist concentration recruits more cells in the population but each cell that responds does so by activating Ca2+ influx in an essentially all-or-none manner. Our results therefore identify a mechanism for eliciting graded responses in a multicellular system despite all-or-none activation of CRAC channels by receptor stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgement
 Disclosures
 References
 
Cell culture

Rat basophilic leukemia cells stably expressing the muscarinic type 3 receptor were a gift from Dr. J. Putney (National Institute of Environmental Health Sciences, Research Triangle Park, NG). Cells were cultured (37°C, 5% CO2) in DMEM with 10% FBS, 2 mM L-glutamine, penicillin-streptomycin, and 100 µM G-418, as described previously (12, 15). For Ca2+ imaging and patch clamp experiments, cells were passaged (using trypsin) onto glass coverslips and used 36–72 h after plating.

Isolation of rat peritoneal mast cells

The mast cells were isolated from female, Sprague-Dawley rats, weighing ~300 g. The animals were sacrificed according to "Schedule 1", (carbon dioxide overdose and neck dislocation). Immediately, 100–150 ml of sterile HEPES buffer (in mM: NaCl 150, KCl 5.6, HEPES 10, NaOH 1.5, MgCl2 1, CaCl2 2, glucose 10 g/l BSA (pH 7.4)) was injected into the peritoneal cavity. The abdomen was massaged for 2 min and then the buffer removed. This was centrifuged at 200 x g for 10 min. The pellet was resuspended in DMEM, triturated, and plated onto glass coverslips (for Ca2+ imaging and immunocytochemistry) or cell-culture dishes (for LTC4 measurements).

Ca2+ imaging

Ca2+ imaging experiments were conducted using the IMAGO CCD camera-based system from TILL Photonics, as described previously (12). Cells were alternately excited at 356 and 380 nm (20-ms exposures) using a Polychrome Monochromator and images were acquired every 2–3 s. Images were analyzed offline using IGOR Pro. Cells were loaded with Fura 2-AM (2 µM) for 40 min at room temperature in the dark and then washed three times in standard external solution of composition (in mM) NaCl 145, KCl 2.8, CaCl2 2, MgCl2 2, D-glucose 10, HEPES 10 (pH 7.4) with NaOH. Cells were left for 15 min in the dark to allow further deesterification. Ca2+ and Ba2+ signals are presented as the fluorescence ratio (356/380) {Delta}R.

Immunocytochemistry

After treatment with carbachol, the cells were fixed in 4% paraformaldehyde in phosphate buffer, for 30 min at room temperature. All washes used 0.01% PBS, (PBS in mM: NaCl 137, KCl 2.7, Na2HPO4 8, KH4PO4 1). The cells were blocked with 2% BSA and 10% goat serum for 2 h. Translocation of protein kinase C{alpha} was visualized using a polyclonal rabbit IgG Ab, (Santa Cruz Biotechnology). After blocking, the cells were washed and incubated in antiPKC{alpha} (1/1000 in 0.2%BSA, 1% goat serum), overnight at 4°C. The cells were thoroughly washed and the secondary Ab (Alexofluor 568-conjugated goat anti-rabbit IgG) was applied at 1/2000 in PBS for 2 h at room temperature. The cells were mounted in Vectastain mounting medium. Images were obtained using a Leica confocal microscope and analysis of translocation conducted using National Institutes of Health image software.

Patch clamp recordings

Whole cell patch clamp experiments were conducted as described (12, 15). Sylgard-coated, fire-polished patch pipettes filled with a solution that contained 145 mM cesium glutamate, 8 mM NaCl, 1 mM MgCl2, 2 mM Mg-ATP, 10 mM HEPES, 10 mM EGTA, 4.6 mM CaCl2 (pH 7.2) with CsOH. Free Ca2+ was buffered at ~140 nM. Pipette resistance was ~5 MOhms when placed in an external solution containing 145 mM NaCl, 2.8 mM KCl, 10 mM CaCl2, 2 mM MgCl2, 10 mM CsCl, 10 mM D-glucose, 10 mM HEPES (pH 7.4) with NaOH. A correction of + 10 mV was applied for the subsequent liquid junction potential that arose from the glutamate-based pipette solution. ICRAC was measured at –80 mV from voltage ramps (–100 to + 100 mV lasting 50 msec) applied at 0.5 Hz, from a holding potential of 0 mV and normalized to cell size (capacitance). Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitized at 100 µs. Capacitative currents were compensated before each ramp. Leak currents were obtained by averaging two ramp currents taken just before application of carbachol and subtracting this from all subsequent recordings. Series resistance was <10 MOhms.

Preparation of cell lysates

Attached cells from 10-cm plastic dishes were washed twice with PBS and lysed with PBS buffer containing 0.5% Triton X-100 and protease mixture inhibitor (Sigma-Aldrich), as described (12). Lysates were centrifuged at 8000 rpm for 5 min, and the supernatants were collected and stored at –70°C until used.

Western blotting

Total cell lysates (40 µg) were analyzed by SDS-PAGE on a 10% gel. Membranes were blocked with 5% nonfat dry milk in PBS plus 0.1% Tween 20 (PBST) buffer for 2 h at room temperature. Membranes were washed with PBST three times and then incubated with primary Ab for 1 h at room temperature. Anti-phospho-ERK Ab, which recognizes dual phosphorylated (i.e., active) ERK, was from New England BioLab and used at 1/2000 dilution. Total ERK 2 Ab was from Santa Cruz Biotechnology and used at a dilution of 1/5000. Secondary Ab was rabbit IgG (1/2500). The membranes were then washed with PBST again and incubated with 1/2000 dilution of peroxidase-linked anti-mouse IgG from Amersham Biosciences for 1 h at room temperature. After washing with PBST, the bands were detected by an ECL-plus Western blotting detection system (Amersham Biosciences).

[3H]Arachidonic acid release

Cells were relabelled with 0.25 µCi/ml [3H]arachidonic acid in DMEM for 1.5 h at 37°C, as described previously (12). Cells were then washed twice with serum-free DMEM to remove unincorporated [3H]arachidonic acid. Thapsigargin with 0.5% fatty acid-free BSA was added for different times (see text). Medium was collected and centrifuged at 25g for 5 min to remove any floating cells. Radioactivity in the supernatant was measured. The amount of [3H]arachidonic acid released into the medium was expressed as a percentage of the total [3H]arachidonic acid uptake.

LTC4 measurements

Following stimulation of attached cells with carbachol, the supernatant was collected and LTC4 levels were measured by enzyme immunoassay (Cayman Chemicals) as described previously (12). In brief, 50 µl of supernatant, leukotriene C4 acetylcholinetransferase and leukotriene C4 antiserum were added to each enzyme immunoassay well. Following incubation for 18 h at room temperature, the wells were emptied and rinsed five times with enzyme immunoassay washing buffer. Two hundred microliters of Ellman’s reagent (prepared fresh) was added to each well and the plate was placed on an orbital shaker for 1.5 h in the dark. Plate absorbance was measured at a wavelength of 405 nm. Data is presented relative to basal LTC4 secretion from nonstimulated cells.

Statistical analysis

Data is presented as the mean ± SEM. Statistical significance was considered as p < 0.01, using Student’s t test, and is denoted by an asterisk (*).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgement
 Disclosures
 References
 
Ca2+ influx following muscarinic receptor stimulation activates cPLA2 and LTC4 production via recruitment of the ERK pathway

Stimulation of cell surface muscarinic receptors with 100 µM carbachol (a maximally effective concentration) raised cytoplasmic Ca2+ (Fig. 1A). In the absence of external Ca2+, carbachol elicited a rapid, albeit transient, rise in cytoplasmic Ca2+ that reflected Ca2+ mobilization from intracellular stores (labeled 0 Ca2+ in Fig. 1A). In the presence of external Ca2+ however, the initial Ca2+ rise developed into a plateau that declined only slowly with time (Fig. 1A). This Ca2+ influx component was central to the activation of cPLA2 because arachidonic acid generation only occurred following stimulation of muscarinic receptors in the presence of external Ca2+ (Fig. 1B). Despite robust and rapid Ca2+ release, carbachol failed to activate cPLA2 in the absence of external Ca2+ (Fig. 1B). The extent of arachidonic acid release following muscarinic receptor activation was not additive with that seen following stimulation with the SERCA pump inhibitor thapsigargin (2 µM for 8 min), which empties stores and maximally activates CRAC channels (Fig. 1B). Arachidonic acid released by cPLA2 activity is subsequently metabolized by the 5-lipoxygenase enzyme to form the potent proinflammatory paracrine signal LTC4 (12). Muscarinic receptor stimulation in the presence, but not absence, of external Ca2+ resulted in secretion of LTC4 and to an extent similar to that seen with thapsigargin (Fig. 1C). Again, there was no additivity between the two stimuli.


Figure 1
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  		FIGURE 1. Ca2+ influx following muscarinic receptor stimulation activates cPLA2 and LTC4 secretion via recruitment of ERK. A, Stimulation with 100 µM carbachol results in Ca2+ release followed by Ca2+ influx into the cells (averaged data from 63 cells is shown). In the absence of external Ca2+, only the Ca2+ release phase is seen (55 cells). B, Carbachol (100 µM) activates cPLA2 and arachidonic acid release but only in the presence of external Ca2+. Arachidonic acid release in response to carbachol is similar in extent to that seen with thapsigargin, is not additive with thapsigargin and is blocked by the MEK inhibitor U0126 (10 µM; pretreated for 15 min). The abbreviation carb. refers to carbachol. C, Carbachol (100 µM) activates LTC4 secretion, but only in the presence of external Ca2+. As with arachidonic acid release, carbachol-evoked LTC4 is similar in extent to that induced by thapsigargin, is not additive with thapsigargin and is suppressed by U0126. D, Muscarinic receptor activation (100 µM carbachol for 8 min) stimulates ERK phosphorylation, and to an extent similar to that seen following challenge with thapsigargin (2 µM for 8 min). ERK phosphorylation to carbachol was prevented by U0126.

 
Stimulation with carbachol in the presence of external Ca2+ triggered phosphorylation (and hence activation) of the mitogen activated protein kinases ERK1 and 2 (Fig. 1D). Application of carbachol to wild-type RBL cells not transfected with muscarinic receptor failed to evoke any resolvable ERK activation (data not shown). Carbachol activated ERK to an extent similar to that seen following stimulation with thapsigargin (Fig. 1D). ERK1/2 are activated following dual phosphorylation of critical threonine and tyrosine residues by the upstream mitogen activated protein kinase kinases MEK1/2. The MEK1/2 inhibitor U0126 (19) suppressed ERK activation following muscarinic receptor stimulation (Fig. 1D). U0126 also suppressed cPLA2 activation (Fig. 1B) and generation of LTC4 (Fig. 1C) following carbachol stimulation. U0126 has no effect store-operated Ca2+ signals (13). Collectively, these results demonstrate that Ca2+ influx following stimulation of muscarinic receptors recruits the ERK pathway, which then results in the generation of the second messenger arachidonic acid as well as the paracrine signal LTC4.

Tight temporal correlation between ERK stimulation, cPLA2 activation, and LTC4 secretion

Fig. 2A shows that stimulation with carbachol (100 µM) resulted in a time-dependent activation of ERK (upper panel) and densitometric analysis of the gel is plotted in Fig. 2B. Generation of arachidonic acid and secretion of LTC4 also increased with the duration of carbachol stimulation (Fig. 2, C and D, respectively). We normalized the extent of ERK activation, arachidonic acid generation, and LTC4 secretion to the value measured after 12 min stimulation and the corresponding time courses are superimposed in Fig. 2E. All three parameters showed similar kinetics of development, consistent with a tight temporal correlation between them.


Figure 2
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  		FIGURE 2. Time course of ERK activation, arachidonic acid release and LTC4 secretion following stimulation with carbachol. A, Western blots showing the extent of ERK phosphorylation after carbachol challenge (100 µM) for the times indicated. Upper panel depicts ERK1/2 phosphorylation and the lower panel depicts total ERK2, used as a control for constant gel loading. B, Data from three experiments as in A is summarized. C and D, Time course of arachidonic acid release (C) and LTC4 secretion (D) are shown. E, Time courses of ERK activation, arachidonic acid release and LTC4 secretion are superimposed to show the close temporal association.

 
Graded dependence on stimulus intensity

To establish the relationship between ERK stimulation, cPLA2 activation, and LTC4 secretion with stimulus intensity, we examined their extent of activation following different levels of muscarinic receptor occupancy. Fig. 3A shows that increasing agonist concentration resulted in graded activation of ERK in cell population measurements. The corresponding dose-response curve is plotted in Fig. 3B. Activation of cPLA2 (Fig. 3C) and secretion of LTC4 (Fig. 3D) were also graded with stimulus intensity. Normalized responses to ERK stimulation, cPLA2 activation, and LTC4 secretion are superimposed in Fig. 3E. Importantly, the curves were superimposable, indicating that each pathway was activated in a graded manner by carbachol and to similar relative extents.


Figure 3
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  		FIGURE 3. Graded activation of ERK, arachidonic acid and LTC4 secretion following stimulation with different concentrations of carbachol. A, Western blots showing the relationship between agonist concentration and ERK phosphorylation. B, Aggregate data from three experiments is summarized. C and D, Corresponding dose-response curves for arachidonic acid release and LTC4 secretion. E, Dose-response curves for the three parameters are superimposed to show the similar dependence on agonist concentration.

 
The receptor-activated Ca2+ influx pathway that drives ERK stimulation involves CRAC channels

The finding that ERK activation, cPLA2 stimulation, and LTC4 secretion, which are all dependent on Ca2+ influx, were graded with agonist concentration is unexpected because agonist-dependent activation of ICRAC by InsP3 is essentially an all-or-none process (15). We considered various possibilities for this apparent discrepancy. For example, receptor activation could recruit additional Ca2+ entry pathways to ICRAC and these could be activated in a graded manner. However, three pieces of evidence militate against this. First, whole cell patch clamp recordings demonstrated that carbachol application activated a nonvoltage-gated inwardly rectifying Ca2+ current with a reversal potential >+60 mV (Fig. 4A), which was indistinguishable from ICRAC activated by store depletion (thapsigargin or dialysis with 10 mM EGTA; Ref. 20). Second, mitochondrial depolarisation suppresses store-operated Ca2+ entry and this reflects, at least in part, enhanced Ca2+-dependent inactivation of CRAC channels due to the loss of mitochondrial Ca2+ buffering (21). Depolarisation of mitochondria with either the complex III respiratory chain inhibitor antimycin A (5 µg/ml) or collapse of the proton gradient with 2mM p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (both in the presence of oligomycin) suppressed carbachol-evoked Ca2+ influx, but without affecting Ca2+ release from the stores (22). Importantly, mitochondrial depolarization suppressed activation of cPLA2 (data not shown) and LTC4 secretion (Fig, 4B) following stimulation with carbachol. Finally, ICRAC is suppressed by application of the CRAC channel blocker 2-aminoethyldiphenyl borate (2-APB) (40 µM; Ref. 2). 2-APB suppressed Ca2+ influx following stimulation with 100 µM carbachol (data not shown) and inhibited subsequent cPLA2 activation (Fig. 4C) and LTC4 secretion (Fig. 4D). We also considered the possibility that receptor stimulation might recruit different Ca2+ influx pathways in a concentration-dependent manner. It has been suggested in HEK293 cells for example that low concentrations of agonist recruit a nonstore-operated Ca2+ influx pathway gated by arachidonic acid that is 2-APB insensitive whereas higher agonist concentrations activate store-operated Ca2+ influx (23). However, 2-APB suppressed Ca2+ influx and both Ca2+-dependent arachidonate release and LTC4 secretion following stimulation with a low concentration of carbachol (1 µM; Fig. 4, C and D), suggesting that Ca2+ influx even at low agonist concentrations in mast cells is via CRAC channels.


Figure 4
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  		FIGURE 4. Graded responses to carbachol despite all-or-none activation of ICRAC. A, Carbachol (100 µM) activates ICRAC. Inset shows the current-voltage relationship taken at steady state. B, Carbachol-evoked LTC4 secretion is impaired by depolarising mitochondria with antimycin A or FCCP (both with oligomycin) but not by oligomycin alone. C and D, The CRAC channel blocker 2-APB suppresses arachidonic acid release (C) and LTC4 secretion (D) to a submaximal (1 µM) and maximal (100 µM) dose of carbachol. E, Ba2+ entry rate through CRAC channels is graded with agonist concentration when all cells are analyzed. F, The fraction of cells that respond to carbachol increases with agonist concentration. Carbachol was applied in Ca2+-free solution and the response measured was Ca2+ release from the stores. G, Ba2+ entry rate is independent of agonist concentration (above threshold) when only those cells that responded in panel F are analyzed. H, ICRAC activation is steeply related to carbachol concentration.

 
Divalent cation entry following receptor stimulation is an apparent all-or-none process

To see whether store-operated Ca2+ influx was supralinearly related to agonist concentration in intact cells, we applied different concentrations of carbachol in Ca2+-free solution to fura 2-loaded cells and then added Ba2+ to the extracellular solution. Ba2+ permeates CRAC channels but, unlike Ca2+, is not transported out of the cytoplasm by Ca2+ATPases and is therefore used as an indicator of unidirectional divalent cation entry (2). We measured the initial rate of Ba2+ entry as an indicator of the number of open CRAC channels and aggregate data from all cells used is summarized in Fig. 4E (n > 150 cells per point). The relationship between carbachol concentration and the rate of Ba2+ entry was clearly graded and correlated well with corresponding agonist-response curves in Fig. 3. However, this dose-response curve reflects all cells in the population i.e., those that respond to carbachol as well as those that do not. Importantly, when we applied carbachol in Ca2+-free solution, taking Ca2+ release as an indicator of receptor activation, we found that only a fraction of the cells responded to lower concentrations of agonist (Fig. 4F). Increasing carbachol concentration increased the number of cells that responded. The graded response for Ba2+ entry in Fig. 4E closely resembles that for agonist responsiveness in Fig. 4F, and therefore is not a true representation of Ba2+ influx. We therefore analyzed the rate of Ba2+ entry only for those cells that responded to carbachol. Results are shown in Fig. 4G. The rate of Ba2+ entry was now independent of agonist concentration for all responding cells. Although less straightforward to interpret due to cytoplasmic Ca2+ removal processes, we also compared the rate of Ca2+ entry following stimulation with low and high concentrations of carbachol. The rate of Ca2+ entry to 1 µM carbachol was 84 ± 9% that seen in 100 µM carbachol and the small difference was not statistically significant (p > 0.3). If each cell responds to a given agonist concentration in an all-or-none way, then the fractional response in a cell population should be equal to the number of cells that are activated. Consistent with this, whereas virtually all cells respond to 100 µM carbachol, ~40% of the cells do so to 1 µM agonist (Fig. 4F). Relative to 100 µM carbachol, 1 µM activates ERK, cPLA2, and LTC4 to similar extents (39.7, 29.5, and 41.0%, respectively; Fig. 3).

Hence, the graded relationship between agonist concentration and divalent cation influx in Fig. 4E is misleading because it represents the likelihood of a cell responding to carbachol in an all-or-none manner rather than graded cation influx into each cell.

To examine this further, we constructed a carbachol dose-response curve for ICRAC activation (Fig. 4H). The relationship was supralinear, consistent with a previous report in which adenosine receptors were activated (15). Increasing agonist concentration increased the number of cells that responded to carbachol but, even at lower agonist concentrations, if a cell responded to carbachol it did so by generating maximal ICRAC. With 1 µM carbachol, 5 of 9 cells responded but for the cells that did, ICRAC was –1.9 ± 0.4 pA/pF. With 100 µM carbachol, in contrast, 10 of 11 cells responded and ICRAC had a similar amplitude (–2.3 ± 0.2 pA/pF; p > 0.1). No ICRAC developed following application of 0.1–0.2 µM carbachol (Fig. 4H), and such concentrations failed to evoke detectable ERK activation, arachidonic acid generation, or LTC4 secretion (Fig. 3).

Muscarinic receptor-evoked responses in acutely isolated rat peritoneal mast cells

The preceding results were obtained using cultured mast cells, raising the question of whether the findings are of physiological relevance. To examine this, we isolated peritoneal mast cells from rats and then designed experiments to see whether varying agonist concentration activated cells in a nonlinear way.

We first explored the classical Ag-IgE-FC{epsilon}RI pathway, which links into the InsP3 signaling cascade. However, responses to Ag are extremely variable in primary mast cells and even a maximal agonist concentration evokes a diverse pattern of Ca2+ signals. Indeed, we observed a range of responses to a fixed Ag concentration, including a transient Ca2+ spike, sinusoidal Ca2+ oscillations, and a sustained Ca2+ signal. Stimulation in Ca2+-free solution followed by Ca2+ readmission failed to trigger a consistent Ca2+ influx signal, with some cells responding by generating Ca2+ oscillations, others a Ca2+ plateau, and some not responding to Ca2+ readmission at all. With such variability, we were unable to dissect out the Ca2+ influx component and hence we turned to other agonists.

Stimulation of muscarinic receptors with 100 µM carbachol resulted in the generation of cytoplasmic Ca2+ signals consisting of Ca2+ release followed by store-operated Ca2+ influx (Fig. 5A), which unlike Ag could be easily separated. Lowering agonist concentration to 10 or 1 µM evoked similar patterns of response, although the percentage of cells responding fell (70% for 1 µM carbachol compared with 99% for 100 µM agonist). Muscarinic receptor stimulation also resulted in graded LTC4 secretion (Fig. 5B). To examine the relationship between agonist concentration and cell responsiveness at the single cell level, we used protein kinase C{alpha} translocation to the plasma membrane as an indicator of cell activation for two reasons. First, translocation of the kinase is a key early step for cPLA2 activation and subsequent LTC4 secretion and hence is of considerable physiological significance. Second, we were able to quantify protein kinase C{alpha} movement with confidence because the mAb we used was very specific. Down-regulation of protein kinase C{alpha} (following overnight exposure to phorbol ester) resulted in complete loss of staining (data not shown). As with RBL cells, stimulation of CRAC channels following exposure to thapsigargin resulted in robust migration of protein kinase C{alpha} to the plasma membrane in primary peritoneal mast cells (Fig. 6A). Images were analyzed on a cell-by-cell basis and aggregate data is summarized in Fig. 6B. We then applied different concentrations of carbachol for four minutes and measured the extent of protein kinase C{alpha} translocation. Histograms were constructed and the pattern of response between control (nonstimulated) cells (Fig. 6C) and those exposed to 1, 10, and 100 µM carbachol compared (Fig. 6, D–F). Compared with the control response profile, raising the carbachol concentration increased the frequency of observing large responses. Aggregate data from all cells is plotted as a dose-response curve in Fig. 6G, and includes cells that responded and those that did not. When all cells are included, the relationship is clearly graded. Fig. 6H plots the data in another way. In this study, we removed all those cells that responded by generating maximal protein kinase C{alpha} translocation (≥95% of response seen to 100 µM carbachol) and then averaged the remaining responses. There was no significant difference between control cells and those exposed to 1 or 10 µM carbachol. Hence, raising carbachol concentration recruited more cells but each cell that responded did so maximally.


Figure 5
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  		FIGURE 5. Muscarinic receptor stimulation evokes cytoplasmic Ca2+ signals and triggers LTC4 secretion in primary rat peritoneal mast cells. A, Typical response to carbachol (100 µM) from a fura 2-loaded primary rat peritoneal mast cell. Carbachol was applied in Ca2+-free solution and then 2 mM Ca2+ was readmitted (arrow). B, Carbachol dose-response curve to LTC4 secretion from primary peritoneal mast cells. *, p < 0.05 and **, p < 0.01 indicate significance relative to control.

 

Figure 6
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  		FIGURE 6. Stimulating muscarinic receptors in rat peritoneal mast cells activates protein kinase C{alpha} in a nonlinear manner. A, Distribution of protein kinase C{alpha} in control (left panel) cells and after stimulation with thapsigargin (2 µM) for 4 min. B, Aggregate data from single cell image analysis from several experiments is shown. C–F, All-points histograms plotting the extent of protein kinase C{alpha} translocation (normalized to the mean response seen in 100 µM carbachol; called % Max. response) against the frequency of observing such a response (expressed as %). C shows the control profile, and D–F the corresponding pattern for 1, 10 and 100 µM carbachol. G, Carbachol concentration is plotted against mean response, with the latter reflecting the responses of all cells. H, The histogram compares signals between control cells and those exposed to 1 and 10 µM carbachol after those cells that responded by evoking protein kinase C translocation ≥95% that of 100 µM carbachol (i.e., x2 sem. from 100%) had been removed from the analysis. Now, the responses are very similar between control, 1 and 10 µM carbachol. Images were analyzed by drawing small rectangles of the same dimensions (4–6 per cell) across the plasma membrane and then computing the fluorescence. Background (taken in regions lacking cells) was subtracted from the data.

 
All-or-none Ca2+ influx following purinoceptor activation in primary mast cells

Mast cells express P2Y receptors (24), which couple to phospholipase C thereby generating InsP3. ATP modulates the responsiveness of mast cells to other stimuli like Ag, increasing the latter’s efficacy in triggering degranulation (24). P2Y receptors desensitize rapidly however, so that the increase in InsP3 levels fall quickly. Ca2+ influx is therefore transient because incoming Ca2+ through CRAC channels is taken up into the stores but in the absence of InsP3, stores refill rapidly and thus deactivate CRAC channels. We therefore used Ba2+ as a surrogate for Ca2+ to probe the extent of CRAC channel activation. Following stimulation (4 min) with different concentrations of ATP in Ca2+-free solution, we applied Ba2+ extracellularly (Fig. 7A) and measured the rate of divalent cation entry. Fig. 7B plots the rate of Ba2+ influx against ATP concentration for all cells exposed to agonist. The relationship is clearly graded. However, as with muscarinic receptor stimulation, not all cells responded to ATP particularly at the lower concentrations. Moreover, even if a cell responded to a low dose of ATP (like 1 µM in Fig. 7A), Ba2+ entry was often barely resolvable and similar to the background Ba2+ influx seen in the absence of agonist. We therefore plotted background-corrected Ba2+ entry only in those cells that generated a Ca2+ release signal to ATP. Aggregate data is summarized in Fig. 7C. Regardless of ATP concentration, if a cell responded it did so in an essentially all-or-none manner.


Figure 7
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  		FIGURE 7. Stimulation of P2Y receptors with ATP activates Ca2+ entry in an all-or-none manner. A, Cells were stimulated with ATP (1 or 100 µM) in Ca2+-free solution and then Ba2+ readmitted. The rate of Ba2+ entry was measured and is plotted against ATP concentration in panel B for all cells (n > 25 cells per point). Because not all cells responded to ATP by generating Ca2+ release, C plots Ba2+ influx rate against ATP concentration only for those cells that responded to ATP by generating Ba2+ entry above resting levels (n > 15 cells per point, except for 1 µM ATP where only one cell responded by evoking Ba2+ influx greater than background levels).

 
The overall extent and kinetics of the Ca2+ signal is sculpted both by Ca2+ release/influx into and Ca2+ removal from the cytoplasm. To see whether different concentrations of ATP affected the rate of removal of Ca2+ from the cytosol, we measured the half-time (t1/2) of decay of the initial Ca2+ transient (in Ca2+-free solution). For 5 µM, 100 µM, and 1 mM ATP, t1/2 of decay was 26 ± 2, 24 ± 1.6, and 28 ± 1.3 s and these values were not significantly different from each other. The amplitude of the Ca2+ transients was similar for the different concentrations. Hence, Ca2+ clearance is not altered by ATP over the range 5 µM –1 mM.

We repeatedly failed to see clear translocation of protein kinase C{alpha} to the plasma membrane over a range of ATP concentrations (data not shown). We attribute this to the transient production of diacylglycerol, due to rapid receptor desensitization. Consistent with this, we did not observe a detectable increase in LTC4 production even at an ATP concentration of 100 µM (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgement
 Disclosures
 References
 
In immune cells, a major route for Ca2+ influx is provided by CRAC channels (18, 25), In mast cells, CRAC channel-dependent Ca2+ influx triggers exocytosis (8) as well as generation of the proinflammatory molecule LTC4 (13). In T lymphocytes, the importance of CRAC channels is underscored by the development of a severe combined immunodeficiency in patients with a mutation in Orai1 (26), a protein that contributes to formation of the CRAC channel pore (26, 27, 28, 29). In this study, we have found that stimulation of two different cell surface receptors in mast cells evokes all-or-none CRAC channel activation. Despite this, graded Ca2+-dependent stimulation of ERK, cPLA2, and LTC4 secretion was obtained in populations of RBL and primary mast cells. This conundrum was resolved by the finding that responses were graded with stimulus intensity because increasing agonist concentration recruited more cells in the population but each cell that responded did so by generating maximal ICRAC and Ca2+ influx. Whereas virtually all cells responded to 100 µM carbachol by developing a cytoplasmic Ca2+ rise and protein kinase C{alpha} translocation to the plasma membrane, ~40% of the cells did so to 1 µM agonist. This proportion correlated well with the functional responses: relative to 100 µM carbachoi, 1 µM carbachol activated ERK, cPLA2, and LTC4 to similar extents (39.7, 29.5, and 41.0%, respectively).

Activation of protein kinase C{alpha} and ERK, which are critical for LTC4 production in mast cells, are largely independent of even large rises in global cytoplasmic Ca2+ (data not shown; W. C. Chang, C. Nelson, J. Di Capite, V. Halse, A. Parekh, submitted), instead being tightly coupled to local Ca2+ influx through CRAC channels (12). Activation of the protein kinase C/ERK/cPLA2 cascade is therefore accomplished primarily through a rise in subplasmalemmal Ca2+ concentration. Because the main determinant of subplasmalemmal Ca2+ concentration is CRAC channel activity, which develops in an all-or-none manner macroscopically, then one would expect significant nonlinearity in the Ca2+-dependent activation of an early downstream target with variations in agonist concentration, and this is indeed seen in the Ca2+-dependent protein kinase C{alpha} translocation experiments of Fig. 6. Nonlinearity here would not necessarily result in all-or-none generation of all downstream products like arachidonic acid and LTC4 though because the intervening metabolic pathways could be subject to regulation by other factors.

Do all-or-none Ca2+ signals contribute to responses in other immune cells? Several studies have reported all-or-none Ca2+ responses in lymphocytes. Interaction of single helper T cells with their physiological ligand, antigenic peptide bound to MHC molecules on APCs, resulted in all-or-none Ca2+ responses and this was independent of Ag concentration (30). As is the case with our results from mast cells, increasing Ag concentration recruited more T cells in the population to respond, but each cell retained its all-or-none Ca2+ response. Cytoplasmic Ca2+ responses to Ag in CTLs are also thought to be an all-or-none process (31). Naive CD4+ T cells generated all-or-none Ca2+ responses following activation by B cells (32). Ca2+-dependent T cell proliferation in response to IL 2 is thought to be an all-or-none process (33, 34). Similarly, phorbol ester-induced activation of the MAP kinase JNK is an all-or-none event (35). Low concentrations of phorbol ester evoke full activation of JNK in a fraction of Jurkat T lymphocytes but no activation at all in the rest of the population. Similarly, all-or-none Ca2+ responses have been observed in neutrophils following stimulation with complement C5a (36) or insoluble immune complex (37).

Despite all-or-none activation of CRAC channels, cells have the ability to produce graded Ca2+ responses. This can be accomplished by varying the membrane potential and therefore electrical driving force for Ca2+ influx or by subsequent inactivation of the CRAC channels by a variety of intracellular signals (reviewed in Ref. 2). Alternatively, nonstore-operated Ca2+ channels could be coexpressed with CRAC channels and gated in a graded manner. In addition to store-operated Ca2+ influx, neutrophils, T and B lymphocytes all express nonstore-operated plasmalemmal Ca2+-permeable pathways including Ca2+-activated Ca2+-permeable nonselective channels (38), TRPM2 (39), TRPV6 (40), and InsP3-gated channels (41). Finally, the cytoplasmic Ca2+ signal will be shaped not only by the rate of Ca2+ entry through CRAC channels but also the rate of Ca2+ removal from the cytoplasm. The latter reflects the concerted actions of plasma membrane transporters, mitochondrial Ca2+ uptake through the uniporter and sequestration into the stores by SERCA pumps (1), all of which are subject to regulation by intracellular signals (42).

In one previous study on human lung-derived mast cells, a low dose of Ag evoked a smaller cytoplasmic Ca2+ rise than a 20-fold higher one (43). It was suggested that Ca2+ signals were thus graded with stimulus intensity rather than being all-or-none events. In that study however, the relative contributions of Ca2+ release and Ca2+ influx to the overall Ca2+ signal were not dissected out. Because low levels of stimulus intensity can trigger some Ca2+ release without subsequent store-operated Ca2+ influx (2, 15), the submaximal concentration of agonist used (43) might not have generated enough InsP3 to ensure stores emptied sufficiently for CRAC channels to activate. Indeed, this was the case with a low concentration of ATP (1 µM) in our experiments (Fig. 7A). Despite clear Ca2+ release, cation entry did not occur. Ca2+ signals to ATP were graded with stimulus intensity because 1 µM ATP evoked only Ca2+ release whereas the higher concentrations triggered both Ca2+ release and influx.

What might be the relevance of our findings to mast cell function? Mast cells are known to secrete strongly and can release their entire secretory contents upon stimulation. Hide et al. (44) have found that ionomycin, a potent activator of CRAC channels through its ability to empty stores rapidly (4), triggers all-or-none degranulation in primary rat mast cells. From a physiological perspective, such a process seems essential because mast cells are generally found in tissues in small numbers and hence need to secrete extensively to affect their immediate environment.


   Acknowledgement
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
Disclosures
 References
 
We thank Dr. Simon Hunt for discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an Medical Research Council Programme Grant (to A.B.P.). W.-C.C. was a recipient of an Overseas Research Studentship award and J.D.C. held a Christopher Welch Scholarship. Back

2 Address correspondence and reprint requests to Prof. Anant Parekh, Oxford University, Parks Road, Oxford, U.K. E-mail address: anant.parekh{at}dpag.ox.ac.uk Back

3 Abbreviations used in this paper: CRAC, Ca2+ release-activated Ca2+; cPLA2, Ca2+-dependent phospholipase A2; LTC4, leukotriene C4; InsP3, inositol 1,4,5-trisphosphate; 2-APB, 2-aminoethyldiphenyl borate. Back

Received for publication June 19, 2007. Accepted for publication August 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 Disclosures
 References
 

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