INTRODUCTION

Although it is generally agreed that an increase in Ca²⁺ is both necessary and sufficient for the initiation of secretion in most excitable cells, the role of Ca²⁺ in nonexcitable cells is less clear (1). The individual Ca²⁺ responses of nonexcitable cells are often extremely heterogeneous, and many models have been proposed for the generation of these complex and often oscillatory patterns (2). Both amplitude-encoded and frequency-encoded Ca²⁺ signals have been proposed (3, 4), and the availability of both mechanisms would allow multiple signaling pathways to be activated by Ca²⁺ in a single cell (5). Hepatocytes, with their repetitive oscillations of constant amplitude but variable frequency are prime candidates for frequency-modulated Ca²⁺ signaling (6), and it has now been shown that mitochondrial NAD(P)H production in these cells is indeed regulated by the frequency of Ca²⁺ oscillations (7, 8). In contrast, it seems clear that the secretory response of single rat salivary acinar cells is tightly coupled to the amplitude of the Ca²⁺ response (9), as is ciliary beating in tracheal epithelial cells (10). Furthermore, it has recently been shown that differential activation of transcription factors in B lymphocytes is achieved via non-oscillatory Ca²⁺ signals of different amplitudes and durations (11).

In most cases, however, it has not been easy to determine the specific features of the Ca²⁺ signal that are important for a physiological response. This is because sensitive methods for detecting function at the single-cell level and with high temporal resolution are not readily available. Furthermore, it now seems clear that additional signals, such as the activation of protein kinase C (12) or one or more G-proteins (13), are often required for the initiation of secretion in nonexcitable cells. We have therefore examined the Ca²⁺ dependence of secretion at the single-cell level using the RBL-2H3 mucosal mast cell. The relatively slow time course of secretion (14) and the complex Ca²⁺ signals seen in response to antigen (5, 15, 16) make this an excellent model system for studying the temporal relationship between Ca²⁺ and exocytosis in nonexcitable cells.

EXPERIMENTAL PROCEDURES

RBL-2H3 mucosal mast cells (17) seeded at low density on glass coverslips were sensitized with dinitrophenyl-specific immunoglobulin E and allowed to incorporate exogenous serotonin as described previously (14). Cells were incubated with 0.5 µM indo-1/acetoxymethyl ester for 20 min at 37 °C in modified Tyrode's solution (135 mM NaCl, 5 mM KCl, 5 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 10 mM Na-HEPES, pH 7.4) containing 250 µM sulfinpyrazone and 0.1% bovine serum albumin (16). Experiments were conducted in modified Tyrode's solution containing 250 µM sulfinpyrazone and 0.05% gelatin. Cells were maintained at 35-37 °C throughout the experiment and were stimulated by perfusing the sample chamber with 3 ml of warm solution containing 1 µg/ml antigen (bovine -globulin to which 10-20 dinitrophenyl groups/molecule had been coupled (18)). Fluorescence of the intracellular Ca²⁺ indicator, indo-1, was excited using light from a Hg lamp filtered at 365 nm. Emitted light from a single cell was split using a 455 nm dichroic mirror and measured with a pair of photomultiplier detectors after filtering at 405 and 485 nm. Data were acquired, stored, and analyzed using Oscar or FeliX software (PTI Inc., South Brunswick, NJ) on a microcomputer equipped with an A/D interface board. The indo-1 ratio ((F₄₀₅/F₄₈₅)  R_min) was calculated after correction for background fluorescence, and the intracellular Ca²⁺ concentration was determined assuming a K_d of 250 nM for indo-1 (19). Microelectrodes for amperometry were prepared by inserting an 8-µm diameter carbon fiber into a glass capillary tube, which was then drawn in a patch pipette puller and sealed with cyanoacrylate glue. Once the glue had cured, the carbon fiber was trimmed back as close as possible (~5 µm) to the glass, and the pipette was back-filled with 3 M KCl. Unless otherwise stated, the electrode potential was set to +550 mV, and the fiber tip was placed <4 µm from the cell surface (20). Amperometric responses were filtered at 500 Hz, digitized, and stored on video tape. Amperometric signals were never detected in unstimulated cells or in cells that had not been loaded with exogenous serotonin (14), thus confirming that they are due to exocytotic release of serotonin. The total amount of serotonin detected was calculated from the integral of the current associated with all amperometric events (21) observed prior to and immediately after detergent lysis for a number of individual cells. From this we estimated that we can detect about 25% of the total cell serotonin (14), which is similar to other estimates for the efficiency of amperometric detection of exocytosis (21, 22).

RESULTS AND DISCUSSION

The response of a typical mast cell when immunoglobulin E receptors on the cell surface are aggregated by antigen is shown in Fig. 1A (16). The Ca²⁺ response was measured using the fluorescent indicator, indo-1 (19), and the release of serotonin from secretory vesicles in the same cell was monitored electrochemically (23) using constant potential amperometry (24). All amperometric events have the same asymmetric shape (Fig. 1B), which is characteristic of exocytotic release of single secretory vesicles (24). As has been shown in other cell types (21, 25), some of the amperometric spikes are preceded by a small rising phase or "foot" (data not shown), which is thought to be due to the slow release of vesicle contents through a narrow fusion pore that forms before full vesicle fusion.

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Secretory events do not occur until intracellular Ca²⁺ is elevated, and in cells where Ca²⁺ is oscillating, amperometric events tend to be clustered around the peaks of the oscillations, and are infrequent or absent in the troughs between oscillations (Fig. 1, A and B). In contrast to pituitary gonadotrophs, where exocytosis is largely confined to the rising phase of a Ca²⁺ oscillation (26), secretory events in the mast cell do not appear to be specifically linked to either the rising or falling phase of the oscillation or to the peak itself (Fig. 1B). There is no obvious threshold concentration of Ca²⁺ for the initiation of secretion, and secretion is not detected during every increase in Ca²⁺ (Figs. 1 and 4). Histogram analysis of the Ca²⁺ concentrations at which secretion is observed in oscillating cells (Fig. 2, narrow hatch marks) shows that the majority of amperometric events occur at intracellular Ca²⁺ concentrations above 300 nM, even though the time spent at such high Ca²⁺ concentrations is relatively small (Fig. 2, wide hatch marks). These findings suggest that the Ca²⁺ signal in mast cells is amplitude-encoded and that an increase in intracellular Ca²⁺ is essential for the initiation of secretion in response to antigen.

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Additional evidence comes from the experiment shown in Fig. 3. After antigen stimulation, intracellular Ca²⁺ increases and secretory events are detected (Fig. 3A). When Ca²⁺ influx is inhibited by depolarizing the cell in high (140 mM) K⁺ (27), intracellular Ca²⁺ drops to resting levels, and secretion is immediately halted. Upon repolarization, intracellular Ca²⁺ increases, and there is a burst of secretion. As intracellular Ca²⁺ falls again, secretion is inhibited but can be restored (Fig. 3B) by stimulating the cell with the Ca²⁺-ATPase inhibitor thapsigargin (28), which depletes stores of Ca²⁺ (29) and stimulates further Ca²⁺ influx (30). Another cycle of K⁺ depolarization and repolarization leads to the same interruption of secretion during the decrease in intracellular Ca²⁺, and when intracellular Ca²⁺ is increased further using the Ca²⁺ ionophore ionomycin, there is another burst of secretion.

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Secretion is rarely seen in cells with oscillations from near resting levels of Ca²⁺, despite peak Ca²⁺ concentrations that match or exceed the levels seen in secreting cells (Fig. 4A). However, when a cell with oscillations from basal Ca²⁺ levels and no secretion progresses to an oscillatory response superimposed on an elevated Ca²⁺ level, secretion begins, even though there is only a small increase in the mean intracellular Ca²⁺ concentration (Fig. 4B). Secretion then increases further during the subsequent high but non-oscillatory Ca²⁺ response (Fig. 4B). Indeed, cells showing sustained, non-oscillatory Ca²⁺ signals in response to antigen usually have excellent secretory responses (Fig. 4C). Secretion is also observed in response to thapsigargin, which elicits a sustained, non-oscillatory increase in intracellular Ca²⁺ (Fig. 4D). Thus, Fig. 4 clearly demonstrates that Ca²⁺ oscillations are neither necessary nor sufficient for exocytosis to occur. Again, this is consistent with an amplitude-encoded Ca²⁺ signaling mechanism, in which a relatively prolonged increase in Ca²⁺ is required for efficient secretion, and argues against a frequency-encoded mechanism (3, 31).

One notable feature of the responses of all cells to antigen is the absence of secretory events during the initial part of the Ca²⁺ response (Figs. 1 and 4). In contrast, when Ca²⁺ influx is briefly interrupted, a burst of secretory events occurs without a delay once influx is restored and intracellular Ca²⁺ increases again (Fig. 3). The relationship between the delay in the onset of the Ca²⁺ response after the addition of antigen (Ca²⁺ lag time) and the delay in the initiation of secretion (secretion lag time) is shown in Fig. 5. As expected, cells with long Ca²⁺ lag times tend to have long secretion lag times, because secretion never occurs unless intracellular Ca²⁺ is increased. There does not appear to be a fixed latent period between the onset of the Ca²⁺ response and the initiation of secretion, but in general secretion occurs 30-60 s after the increase in Ca²⁺ (Fig. 5).

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The initial Ca²⁺ oscillations have been attributed to release of Ca²⁺ from intracellular stores because they do not depend on Ca²⁺ influx (16); influx is, however, necessary to replenish the stores, sustain the Ca²⁺ response (16), and support secretion (27). Thus, a possible explanation for the lack of secretion during the first few oscillations is that secretion does not occur until stores have been depleted sufficiently to activate Ca²⁺ influx. However, using the manganese influx technique to monitor the antigen-induced activation of the Ca²⁺ influx pathway (32, 33), we were unable to dissociate the initial increase in Ca²⁺ from the activation of influx (Fig. 6; n = 5 cells). Thus the delay in secretion cannot be attributed to slow activation of Ca²⁺ influx.

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It is possible that the first few Ca²⁺ oscillations are required for the transport or docking of secretory vesicles (34). In excitable cells, both the recruitment of secretory vesicles and exocytosis itself are Ca²⁺-dependent (34). However, our finding that secretion is immediately halted when intracellular Ca²⁺ is reduced to resting levels but resumes as soon as intracellular Ca²⁺ increases again (Fig. 3) suggests that Ca²⁺-dependent vesicle docking may not be the rate-limiting step in the initiation of secretion from mucosal mast cells. Another possibility is that the initial Ca²⁺ response is necessary for the production of other intracellular messengers (12, 35). A detailed biophysical study of the Ca²⁺ dependence of exocytosis in peritoneal mast cells dialyzed with a nonhydrolyzable analogue of guanosine triphosphate, GTPS,¹ also found little or no secretion during the initial increase in Ca²⁺ (36). It was suggested that this might be due to the slow activation of protein kinase C (36), which is also required for secretion in mast cells (12), and this may also be an explanation for our results. Consistent with this, we found that addition of the protein kinase C activator, phorbol myristate acetate (50 nM), 5-8 min before antigen reduced the delay between the initial increase in Ca²⁺ and the initiation of secretion from 34 ± 17 s (mean ± S.D., n = 9 cells) to 16 ± 8 s (n = 10 cells; p < 0.025, Student's unpaired t test).

It is well established that Ca²⁺ influx is required for antigen-induced secretion in RBL-2H3 mucosal mast cells (27, 37), and Fig. 7A confirms that secretion does not occur until extracellular Ca²⁺ is restored to an antigen-stimulated cell. However, the role played by Ca²⁺ influx is not clear. Because the Ca²⁺ response due to release from stores in the absence of influx is relatively transient and often consists of oscillations to base-line levels of Ca²⁺ (Fig. 7A and Ref. 16), it may be that influx is simply required to sustain the Ca²⁺ response for long enough for secretion to occur. Alternatively, it is possible that microdomains of high Ca²⁺ close to the site of exocytosis, which are known to be important in excitable cells (38), might also occur in mast cells (39). Because the conductance of the calcium release-activated Ca²⁺ channel found in mast cells and lymphocytes is much smaller (40) than that of the voltage-sensitive Ca²⁺ channels found in excitable cells, it may not be possible to generate such steep gradients of Ca²⁺ close to the plasma membrane in mast cells. In contrast, the Ca²⁺ conductance of inositol trisphosphate receptor channels is high (41), so Ca²⁺ microdomains in the vicinity of the stores may be more important for exocytosis in nonexcitable cells. If this is the case, then depleting stores of Ca²⁺ and preventing them from refilling using the Ca²⁺-ATPase inhibitor thapsigargin should prevent secretion even when Ca²⁺ influx is restored. However, as Fig. 7B shows, exocytotic events still occur under these conditions, provided that extracellular Ca²⁺ is present. These results clearly demonstrate that Ca²⁺ influx across the plasma membrane can directly trigger exocytosis and that refilling and continued release of Ca²⁺ from stores and any store-associated microdomains of Ca²⁺ are not essential for exocytosis in mucosal mast cells.

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To our knowledge, this is the first study to examine in detail the temporal relationship between individual secretory events and Ca²⁺ oscillations in nonexcitable cells, and it clearly demonstrates that the Ca²⁺ signal in individual mucosal mast cells is amplitude-encoded. We have also confirmed at the single-cell level that there is an absolute requirement for elevated intracellular Ca²⁺ in antigen-induced secretion from mucosal mast cells and that this secretion depends on Ca²⁺ influx.